INVESTIGATING THE ROLE OF THE ESSENTIAL GTPASE RBGA IN
THE ASSEMBLY OF THE LARGE RIBOSOMAL SUBUNIT IN BACILLUS
SUBTILIS
By
Megha Gulati

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Genetics - Doctor of Philosophy
2014

ABSTRACT
INVESTIGATING THE ROLE OF THE ESSENTIAL GTPASE RBGA IN THE ASSEMBLY
OF THE LARGE RIBOSOMAL SUBUNIT IN BACILLUS SUBTILIS
By
Megha Gulati
Ribosomes are large ribonucleoprotein complexes required for synthesis of proteins and are a
major target of several types of antibiotics. Ribosome assembly defects have been linked to
several diseases, most notably Diamond-Blackfan anemia and Schwachman-Diamond syndrome.
Although ribosome assembly has been studied extensively through in vitro reconstitution
analysis, relatively little is known about the steps involved in vivo. Assembly factors such as
GTPases are a crucial universal requirement for ribosome assembly yet their precise role has
remained elusive. This thesis focuses on characterization of RbgA (ribosome biogenesis GTPase
A), an essential GTPase in Bacillus subtilis, and its role in the assembly of the large ribosomal
subunit. RbgA is widely distributed evolutionarily and its homologs have been functionally
implicated in ribosome assembly in yeast (Lsg1p, Nog2p and Nug1p) as well as mammalian
cells (Mtg1). Analysis of the ribosome assembly intermediate isolated from RbgA-depleted cells
indicated that the GTPase plays a crucial role at a late stage in maturation of the 50S subunit and
in recruitment of r-proteins L16, L27 and L36 during the assembly process. To elucidate the role
of RbgA in 50S assembly, first, we performed extensive biochemical analysis to determine the
kinetic parameters of RbgA and its interaction with the 50S subunit and ribosomal intermediates
+

(Chapter 2). These studies revealed that RbgA requires K ion for optimal GTPase activity and
this activity is enhanced ~60 fold only in the presence of mature 50S subunit, and laid the
groundwork for developing a model for the role of RbgA in the 50S assembly process. Next, we

generated a library of loss-of-function mutant RbgA proteins through site-directed mutagenesis
and performed kinetic analyses and biochemical characterization to delineate the critical
functional sites of the enzyme (Chapter 3). To analyze the mutations in vivo, we engineered B.
subtilis strains to express mutated RbgA protein(s) and analyzed the ribosomal intermediates
formed in the cells. These studies identified a potential catalytic residue of RbgA and provided
insight into its catalytic mechanism when complexed with its ribosomal substrate. Further, we
identified and characterized an RNA-binding domain required for RbgA function. Lastly, we
designed a genetic suppressor screen, and isolated, and characterized six independent extragenic
suppressor mutations that partially alleviated the growth defect in the RbgA-defective B. subtilis
strain (Chapter 4). These studies revealed a novel in vivo ribosomal intermediate and determined
a functional interaction between RbgA and r-protein L6. Together, these studies provide a model
for the molecular mechanism of RbgA interaction with the 50S ribosomal subunit and the role of
the enzyme in the assembly process.

Copyright by
MEGHA GULATI
2014

I dedicate this thesis to my loving family, my father, Pankaj, my mother, Anjali, and my best
friend and sister, Neha, who have supported me and encouraged me to follow my dreams
across the oceans, and to my husband Shiven, whose unconditional love and support
provided me with the strength and perseverance I needed to achieve my goals.

v

ACKNOWLEDGEMENTS
First and foremost I express my gratitude to my mentor, Dr. Robert Britton, for his support and
guidance on my projects and my maturation through graduate school. He has been a wonderful
supervisor, mentor and friend who taught me innumerable scientific and life lessons. He
encouraged me to think critically, be the toughest critic of my own work and helped me hone my
skills as a scientist. His sound advice, insightful criticisms and thoughtful guidance are
invaluable and deeply appreciated. My experience of working under his guidance is one I will
always remember and cherish.

I would also like to thank my committee members: Dr. Lee Kroos, Dr. Robert Hausinger, Dr.
Donna Koslowsky and Dr. Ronald Patterson for their continued advice and support throughout
my projects and scientific career. My acknowledgement also goes to Dr. Lee Kroos for the
discussions during our joint group meetings and I am grateful for the ideas and suggestions that
contributed to the work presented in this thesis. A special thanks for allowing me to use the
chemiluminescence imager. In addition, my thanks to Dr. Barb Sears for her support and
guidance and Jeannine Lee for her tremendous help in solving the administrative issues I came
across and helping me assimilate to a new university system.

My appreciation for all the past and present members of Dr. Britton’s laboratory, for numerous
stimulating discussions and all their camaraderie. They made the lab a fun place to work, and
were a large part of my academic and personal life. My wonderful labmates and friends at MSU;
Caleb, Amy, Hirosha, Nicole, Denise and Anthony; our frequent outings as a group were most

vi

enjoyable and made me feel at home, far away from, India, my home country. They provided me
ceaseless support, necessary ‘distractions’ and lots of laughs.

Lastly, I would like to thank my family, my parents Anjali and Pankaj, for always encouraging
me to pursue my interests and passion, for waking up at odd hours in India so we could talk
according to US time, for moving heaven and earth to plan my dream wedding in Michigan as I
couldn’t travel home and for lots of gift and ‘care packages’ sent through anyone who was
traveling from Delhi to anywhere in the US. Also, I could not have done any of this without the
love and support of my sister Neha, who kept me grounded by reminding me that there exists a
world that doesn’t revolve around me and wisely did not follow in my footsteps choosing instead
to complete her MBA. We shared a room for 19 years, sharing secrets and whispering late into
the night and it was most painful to leave you behind to follow my dreams of research and
pursuit of doctoral work.
My new family, Preeti and Navin Kapur receive my sincere love and gratitude for accepting me
as a second daughter and for loving me unconditionally. I would especially like to acknowledge
Shivani Kapur, who provided me a shoulder to lean on and for being a sister-in-arms in our
shared experience in navigating life and family.
Finally, I would like to thank my loving husband Shiven, for encouraging me to shoot for the
stars, for sharing my passion of science as a fellow scientist and for numerous late night
discussions and troubleshooting problems, for always being there for me and for never letting
geographical distance be a factor in our relationship. Without you all none of this would have
been possible.

vii

TABLE OF CONTENTS

LIST OF TABLES ...................................................................................................................... xi
LIST OF FIGURES ................................................................................................................... xii
CHAPTER 1: Literature Review ............................................................................................. 1
Introduction........................................................................................................................... 2
Bacterial Ribosome ............................................................................................................... 2
Ribosome Assembly in Bacteria ........................................................................................... 3
Assembly of the 50S subunit ......................................................................................... 5
Factors implicated in Ribosome Assembly .......................................................................... 6
RNA Modification Enzymes ......................................................................................... 7
RNA Helicases .............................................................................................................. 7
Chaperone Proteins ........................................................................................................ 9
Bacterial GTPases implicated in Ribosome Assembly ........................................................ 9
GTPases implicated in the Assembly of the 50S subunit ................................................... 10
RbgA............................................................................................................................ 10
ObgE ............................................................................................................................ 12
YsxC ............................................................................................................................ 13
YphC............................................................................................................................ 13
Era................................................................................................................................ 14
RsgA ............................................................................................................................ 15
YqeH............................................................................................................................ 15
Unique features of Ribosome Assembly GTPases ............................................................. 16
HAS-GTPases.............................................................................................................. 17
Circularly permuted GTPases...................................................................................... 17
Affinity for guanine nucleotides .................................................................................. 18
Possible mechanisms by which GTPases could participate in Ribosome Assembly ......... 18
REFERENCES ................................................................................................................... 20
CHAPTER 2: Biochemical characterization of the ribosome assembly GTPase RbgA in
Bacillus subtilis ......................................................................................................................... 28
Abstract ............................................................................................................................... 29
Introduction......................................................................................................................... 30
Materials and Methods ....................................................................................................... 31
Growth conditions and strain construction .................................................................. 31
Purification of RbgA proteins ..................................................................................... 32
Preparation of ribosomal particles ............................................................................... 33
Characterization of GTPase activity of RbgA ............................................................. 34
Stimulation of GTPase activity of RbgA by ribosomal subunits ................................ 35
Importance of potassium ion for GTPase activity of RbgA ........................................ 35

viii

Homology modeling of the K-loop of RbgA .............................................................. 36
Interaction between RbgA and ribosomal particles..................................................... 36
Results................................................................................................................................. 37
RbgA has a low intrinsic GTPase activity ................................................................... 37
The GTPase activity of RbgA is maximally stimulated by mature or free 50S
ribosomal subunits ...................................................................................................... 38
Potassium is required for optimal GTPase activity ..................................................... 39
RbgA interacts with the 50S ribosomes and 45S intermediates in a nucleotide specific
manner......................................................................................................................... 40
Discussion ........................................................................................................................... 41
ACKNOWLEDGEMENTS ................................................................................................ 53
REFERENCES ................................................................................................................... 54
CHAPTER 3: Mutational analysis of the ribosome assembly GTPase RbgA provides
insight into ribosome interaction and ribosome-stimulated GTPase activation ................ 58
Abstract ............................................................................................................................... 59
Introduction......................................................................................................................... 60
Materials and Methods ....................................................................................................... 62
Growth conditions ....................................................................................................... 62
Construction of strains ................................................................................................. 62
Characterization of association between RbgA or mutants and 45S or 50S
complexes ................................................................................................................... 63
Characterization of GTPase activity of RbgA/mutants ............................................... 65
Bioinformatics analysis and superimposition.............................................................. 65
Results................................................................................................................................. 65
Multiple sequence alignment of RbgA homologs revealed 4 regions of high
conservation ................................................................................................................ 65
The C-terminal domain of RbgA contains an ANTAR RNA binding domain ........... 66
Site-directed mutagenesis and phenotypic analysis of RbgA mutants ........................ 67
Biochemical characterization of RbgA mutants .......................................................... 70
Mutations in the ANTAR domain alter RbgA:ribosome association .......................... 70
Mutations in CR1 severely reduce GTPase activity of RbgA ..................................... 72
Linker region is required for RbgA function ............................................................... 74
Strains expressing mutated RbgA proteins do not accumulate a novel intermediate.. 75
Discussion ........................................................................................................................... 75
ACKNOWLEDGEMENTS ............................................................................................. 101
REFERENCES ................................................................................................................. 102
CHAPTER 4: Functional interaction between ribosomal protein L6 and RbgA during
ribosome assembly ................................................................................................................ 107
Abstract ............................................................................................................................. 108
Introduction....................................................................................................................... 109
Materials and Methods ..................................................................................................... 111
Growth conditions ..................................................................................................... 111
Plasmids ..................................................................................................................... 111
Construction of strains ............................................................................................... 112

ix

Suppressor screen ...................................................................................................... 112
Whole genome sequencing and bioinformatics analysis ........................................... 113
Structure analysis....................................................................................................... 114
Construction of strains for determining the phenotype of the L6 protein in a wild-type
background ................................................................................................................ 114
Analysis of ribosome profiles and ribosome complexes ........................................... 115
In vitro maturation ..................................................................................................... 116
GTPase activity ........................................................................................................ 116
Results............................................................................................................................... 117
Construction of a strain containing a rbgA mutation with a growth phenotype suitable
for a genetic suppressor screen ................................................................................. 117
Suppressor mutations localize to the rplF gene, which encodes the ribosomal
protein L6 .................................................................................................................. 118
Suppressor strains accumulate a novel ribosomal intermediate that is distinct from the
45S particle ............................................................................................................... 119
rplF mutations do not impair growth but are partially defective in ribosome subunit
joining ....................................................................................................................... 120
The 44S particle can be matured into a 50S subunit in vitro..................................... 121
Discussion ......................................................................................................................... 122
REFERENCES ................................................................................................................. 149
CHAPTER 5: Summary and significance ........................................................................... 154
Introduction....................................................................................................................... 155
Role of GTPase activity .................................................................................................... 156
Interaction with rRNA ...................................................................................................... 157
RbgA and assembly of the 50S subunit ............................................................................ 158
REFERENCES ................................................................................................................. 160

x

LIST OF TABLES

Table 2.1 Kinetic parameters of RbgA in the presence and absence of ribosomal subunits .... 45
Table 2.2 Intrinsic GTPase activity of RbgA mutants .............................................................. 46
+

+

Table 2.3 GTPase activity of RbgA in the presence of Na and K ions ................................. 47
Table 3.1 List of B. subtilis strains used in this study ............................................................... 81
Table 3.2 Results from a search of structures similar to RbgA C-terminal domain in DALI
server ......................................................................................................................... 84
Table 3.3 Growth rate of RbgA mutants ................................................................................... 85
Table 3.4 Dominant negative phenotype observed under specific growth conditions.............. 86
Table 3.5 Results for in vitro binding of RbgA mutants to the 45S and 50S subunits under
different nucleotide conditions .................................................................................. 87
Table 3.6 Measurement of GTPase activity of RbgA mutants in the presence and absence of
the 50S subunit .......................................................................................................... 88
Table 4.1 List of Strains used in this study ............................................................................. 127
Table 4.2 Suppressor mutations in ribosomal protein L6 ....................................................... 129

xi

LIST OF FIGURES

Figure 2.1 Kinetic analysis of GTP hydrolysis rates by RbgA proteins ................................... 48
Figure 2.2 Stimulation of GTPase activity of RbgA by ribosomal particles ............................ 49
Figure 2.3 Superimposition of MnmE and homology model of RbgA .................................... 50
Figure 2.4 Interaction between RbgA and ribosome in the presence of different guanine
nucleotides ............................................................................................................... 51
Figure 2.5 Proposed model for role of RbgA in 50S subunit maturation ................................. 52
Figure 3.1 Sequence alignment of RbgA with its homologs .................................................... 89
Figure 3.2 C-terminal domain of RbgA contains RNA binding domain ANTAR ................... 92
Figure 3.3 Testing the growth phenotype of RbgA mutants ..................................................... 93
Figure 3.4 Consolidated results of mutational analysis of RbgA ............................................. 94
Figure 3.5 An example of growth curves comparing wild type growth with a mutation that
causes a mild growth defect and a mutation that causes a severe growth defect..... 95
Figure 3.6 In vitro binding assay for RbgA mutants to 50S subunit under different nucleotide
conditions ................................................................................................................. 96
Figure 3.7 A ligand based structural superimposition of EF-Tu over the free RbgA ............... 97
Figure 3.8 A ligand-based superimposition of RbgA and EF-Tu for comparison of catalytic
machinery is shown.................................................................................................. 98
Figure 3.9 Ribosome profiles of RbgA mutants ....................................................................... 99
Figure 3.10 Proteomic analysis of ribosomal intermediate formed in strains expressing RbgA
mutant protein ..................................................................................................... 100
Figure 4.1 Phenotype of RB1043 (rbgA-F6A) and suppressor strains ................................... 130
Figure 4.2 Multiple sequence alignment of L6 protein from selected bacterial species ......... 131
Figure 4.3 Homology model of L6 protein depicting the suppressor mutations .................... 134

xii

Figure 4.4 The ribosome profile of RbgA-F6A suppressor strain show an accumulation of a
novel 44S complex................................................................................................. 135
Figure 4.5 Analysis of ribosomal proteins in 44S intermediates ............................................ 139
Figure 4.6 Analysis of ribosomal protein composition of 44S intermediates ......................... 140
Figure 4.7 Measurement of GTPase activity of RbgA in the presence of 44S intermediate from
suppressor strains ................................................................................................... 141
Figure 4.8 Mutations in L6 protein affect subunit joining/interaction .................................... 142
Figure 4.9 Mutations in L6 protein affect subunit joining/interaction .................................... 143
Figure 4.10 Analysis of ribosomal proteins L6 and L16 in 50S subunits that accumulate in
strains expressing rplF substitutions in wild-type background .......................... 144
Figure 4.11 In vitro maturation of large subunit intermediates .............................................. 145
Figure 4.12 Interactions between L6 protein with the 50S ribosomal subunit ....................... 146
Figure 4.13 Proposed model for the role of RbgA in the mediating productive h97-L6
interactions during the assembly of large ribosomal subunit.............................. 147
Figure 4.14 Conserved interactions between L6 and helix 97 of 23S rRNA (ScL9-h97) ...... 148

xiii

CHAPTER 1
Literature Review

1

Introduction
Ribosomes are large ribonucleoprotein machines that are essential for decoding mRNA to
synthesize proteins [1,2]. Extensive structural and biochemical studies over the last several
decades have provided insights about the function of ribosome and the translational machinery
that regulates [3-6]. In contrast relatively little is known about the in vivo assembly of this
complex. Though functionally active ribosomal subunits can be reconstituted in vitro from its
purified components this process requires extreme non-physiological conditions [7-14]. In
addition, the slow kinetics and low efficiency of in vitro reconstitution strongly suggests that
ribosomal assembly factors are required for in vivo assembly. While more than 200 assembly
factors have been indentified in eukaryotes relatively fewer are known in prokaryotic ribosome
assembly [15-17]. These assembly factors include rRNA modification enzymes, RNA helicases,
chaperone proteins and GTPases [18-23]. In bacteria GTPases comprise the largest class of
essential ribosome assembly factors [22,23]. Several GTPases have been implicated in assembly
of the 50S as well as the 30S subunit [22,23]. Most of these GTPases are widely conserved and
in most instances the homologs of these proteins have also been implicated in ribosome
assembly. In this chapter a general overview of ribosome assembly is discussed with an
emphasis on the assembly factors implicated in ribosome assembly in vivo. Also highlighted are
several GTPases that are involved in the assembly of the 50S and 30S subunits and the unique
features of these enzymes.

Bacterial Ribosome
Ribosomes are essential macromolecular machines that catalyze the fundamental cellular process
of protein synthesis [1-3,6]. In Escherichia coli 40% of the cells energy output is channeled

2

towards ribosome assembly and protein synthesis [18]. Ribosomes are also the targets of most
antibiotics that bind to specific sites on the ribosome and inhibit protein synthesis [24-28]. The
bacterial ribosome is a 70S complex that comprises of two individual subunits, the large subunit
50S and the small subunit 30S [6,29]. The 50S subunit is composed of 23S rRNA, 5S rRNA and
33 ribosomal proteins (r-proteins) designated as L1-L36 and contains the peptidyl transferase
center, the site of peptide bond formation [3,6]. The 16S rRNA and 21 ribosomal proteins
designated as S1-S21 form the 30S subunit which is responsible for binding to the mRNA during
translation and contributes to the fidelity of translation by monitoring base pairing between
codon and anticodon in the decoding process [3,6].
The availability of high resolution crystal structures of the ribosome bound to various antibiotics
have provided a clear picture of the catalytic sites that lie within the rRNA and contributed to a
detailed understanding of the mechanism of translation [3-6]. Each subunit has three binding
sites for tRNA, the A-site (aminoacyl) that accepts the aminoacyl tRNA, the P-site (peptidyl) that
is bound to the tRNA with a nascent peptide chain and the E-site (exit) which holds the
deacylated tRNA before it exits the ribosome [2,30]. These sites are crucial to the function of the
ribosome and are the target of most antibiotics that bind to the ribosome and inhibit protein
synthesis [24,31]. However the details of how these complex structures are assembled in vivo via
the precise and coordinated binding of >50 r-proteins and rRNA into a functional
multicomponent complex is lacking.

Ribosome Assembly in Bacteria
Ribosome assembly is a complex and tightly regulated process that involves the coordinated
assembly of >50 r-proteins and 3 rRNA molecules to form functional subunits [1]. In actively

3

growing bacteria 40% of total energy production is consumed by ribosome assembly and thus
successful and efficient ribosome assembly is critical for the cells [9]. Ribosome assembly
defects have been linked to several diseases including cancer and diabetes [32]. Several cancers
show a deregulation of the ribosome assembly and function most notably Diamond-Blackfan
anemia (DBA), Schwachman-Diamond syndrome and tuberous sclerosis [33,34]. DBA is a
congenital bone marrow failure syndrome characterized by red blood cell aplasia, macrocytic
anemia and increased risk of malagnancy and mutations in four r-proteins S19, S24, S17 and
S35a is seen in 30% of DBA patients [34]. In addition mutations in r-proteins L5 and L11 are
associated with cleft palate and physical abnormalities including thumb and heart anomalies
[35]. While it is clear that mutations in r-proteins disrupt ribosome biogenesis and cause specific
clinical phenotypes how these mutations lead to the disease state is unknown. R-proteins are
some of the most conserved proteins across all kingdoms of life and their relative position and
structure in the ribosomes is similar in prokaryotic and eukaryotic cells [6,29]. Understanding the
role of r-proteins in ribosome assembly in vivo in bacterial cells would provide insight into the
role of their homologs in eukaryotic cells and help elucidate the principles underlying these
ribosomopathies and may also lead to new therapeutic strategies [34,36].
Ribosome assembly is an intricate process and the basic steps involve a) the transcription,
processing and modification of rRNA; b) the proper folding of rRNA into distinct domains; c)
the translation and modification of r-proteins; d) the binding of r-proteins to rRNA in a precise
manner and e) the binding and release of various assembly factors involved at different steps in
the process. Many of these steps are coupled and occur in parallel in the cell.
In the late 1960s Nomura and coworkers demonstrated the assembly of an active 30S subunit
from free rRNA and r-proteins without any additional factors [11,37]. Successful in vitro

4

reconstitution of the 50S subunit was soon achieved by Nierhaus and Dohme [38]. This
pioneering work highlighted the fact that all the information necessary for ribosome assembly is
encoded within the rRNA and r-proteins themselves [9]. The ability to reconstitute active
ribosomal subunits represented a major breakthrough as it became possible to dissect ribosome
assembly by altering the various components involved in the process. These crucial experiments
led to the construction of assembly maps for both the 30S and the 50S subunits [39,40].

Assembly of the 50S subunit
In vitro assembly of the 50S subunit in Escherichia coli is a four step process that involves three
intermediates [7,38,41]. The first intermediate RI50(I) migrates at 33S and contains the 23S
*

rRNA, 5S rRNA and 22 r-proteins. Conversion of RI50(I) to the second intermediate RI50 (I)
that migrates at 41S-43S requires an extended incubation at high temperature. This is followed
by the addition of the remaining 11 r-proteins to form the final intermediate RI50(2) which
migrates at 48S and contains all the components of an active 50S subunit. However, a further
incubation step at high temperature and under high ionic conditions is required to form the
mature 50S subunit.
Reconstitution of 50S subunits in Bacillus stearothermophilus reveals that in vitro assembly is
distinct from that seen in E. coli resulting in a different intermediate during the assembly process
[13,42]. The major intermediate in B. stearothermophilus is RI50 which lacks only three rproteins, the identities of which were not determined. Addition of the missing proteins and a long
incubation at high temperatures yields a functional 50S subunit.

5

Aside from a few studies in B. stearothermophilus and recent studies in Bacillus subtilis the bulk
of our knowledge on ribosome assembly stems from research in E. coli [22]. The prevailing idea
was that a single pathway or regulatory mechanism for ribosome synthesis and assembly existed
and E. coli was the chosen representative of bacteria. However, mounting evidence now
indicates that the majority of bacteria likely utilize different mechanism and regulatory systems
for ribosome synthesis compared to E. coli [18,22,41]. This underscores the need to investigate
the ribosome assembly pathways and regulatory apparatus in other systems such as B. subtilis
that have largely been ignored.

Factors implicated in Ribosome Assembly
While in vitro reconstitution of ribosomal subunits requires 90 minutes ribosomes are assembled
in the cell at 37°C in two minutes [43]. The harsh non-physiological conditions such as long
incubation times, high ionic concentrations and high temperature required for in vitro ribosome
assembly suggested that additional factors must be involved in the assembly process in vivo. In
the past decade there has been a large increase in the identification of assembly factors in
bacteria as well as eukaryotes [41,43,44]. While more than 200 assembly factors have been
identified in eukaryotes relatively few proteins in bacteria have been shown to be involved in
ribosome assembly [15,45]. These factors include GTPases, RNA helicases, chaperone proteins
and rRNA modification factors [18,44]. Characterization of assembly factors in bacteria will
elucidate the mechanistic details of how these proteins assist in ribosome assembly. In addition
these assembly factors represent novel targets for modern antibacterial drug discovery and could
be part of a solution to multi-drug resistance in bacteria [46,47].

6

RNA Modification Enzymes
Ribosome assembly begins with the transcription of rRNA in a single primary transcript which is
processed by a number of endonucleases [48,49]. Modification of rRNA occurs posttranscriptionally and consists of two main types, methylation and pseudo-uridinylation. While
modification of rRNA is conserved in all organisms the site of modifications and the number of
modifications varies. Mapping of all methylations and pseudouridinylations onto the ribosome
reveal that they cluster around active sites of the ribosome such as the tRNA-mRNA binding site
and peptidyltransferase center [18]. However, the functional role of these modifications remains
a mystery [18]. Some rRNA modifications have been linked to antibiotic resistance indicating
that these studies could result in findings relevant to study and design of drug targets [24]. While
several pseudouridine synthases and methyltransferases have been identified by deletion analysis
only rluD has been shown to play a role in ribosome assembly [21]. RluD is a pseudouridine
synthase required for ribosome assembly in E. coli [21]. This enzyme acts on the 23S rRNA and
loss of RluD results in immature 50S subunits and a breakdown of unstable 70S subunits [21].

RNA Helicases
rRNA is prone to the formation of unfavorable secondary structures and thus RNA helicases play
an important role in ribosome assembly [50,51]. A large subfamily of RNA helicases are termed
DEAD box helicases as they contain conserved motif Asp-Glu-Ala-Asp (DEAD) [19,52]. They
are conserved from bacteria to viruses to humans and these proteins are ATP-dependant
[19,52,53]. These proteins are believed to play multiple roles in ribosome assembly as they are
needed for helicase activity, unwinding of local RNA secondary structure, act as RNA
chaperones by assisting with proper RNA folding and modulate RNA-protein interactions [41].

7

While DEAD box proteins are essential for eukaryotic ribosome assembly they are dispensable
for assembly in bacteria under normal growth conditions [20]. Only three DEAD box helicases
SrmB, CsdA and DbpA have been implicated in assembly of the 50S subunit in E. coli
[19,20,54]. CsdA is induced by cold shock suggesting that the enzyme may be important under
stress conditions [20]. Deletion of wither csdA or srmB leads to a temperature sensitive
phenotype likely due to rRNA misfolding at low temperatures [19]. Deletion of csdA results in
an immature 50S complex that lacks late assembly proteins. In contrast the deletion of srmB
results in accumulation of a pre-50S complex that lacks the early assembly r-protein L13. Studies
have shown that the deletion of csdA and srmB suppress temperature sensitive mutations in
genes encoding for r-proteins S2 and L24 respectively [19,20].
DbpA differs from CsdA and SrmB in that it requires a specific sequence present in helix 92 of
23S rRNA for its helicase activity [20,54]. Studies of DbpA homolog in B. subtilis YxiN indicate
that the region responsible for specificity is located in the C-terminal of the protein [55].
Interestingly the deletion of dbpA does not result in a growth defect [20]. However, the
expression of a DbpA mutant yields a dominant slow growth phenotype and results in defects in
ribosome assembly [54]. In this mutant the maturation of 50S subunits is affected resulting in
accumulation of immature particles. This suggests that the presence of an inactive or partially
active DbpA disrupts assembly of the 50S subunit while complete absence of the protein does
not affect assembly. Taken together these studies suggest that helicases play a role in ribosome
assembly under stress conditions such as low temperature however the mechanistic details of
their role in assembly process is unknown.

8

Chaperone Proteins
Another set of protein factors that have been implicated in ribosome assembly are chaperone
proteins [56,57]. While RNA helicases seem to be needed at low temperatures for proper
ribosome assembly, chaperone proteins play an important role in biogenesis at high
temparatures. The chaperones DnaJ and DnaK are part of heat shock protein 70 (HSP70)
chaperone machine DnaK-DnaJ-GrpE and have been implicated in ribosome assembly [56].
DnaK has been shown to stably associate with pre-30S complex and it has been proposed that
DnaK overcomes the high temperature activation step required for the in vitro reconstitution of
30S subunits. Strains with a deletion in dnaK gene show no assembly defects at lower
temperatures (30°C) but at temperature above 42°C ribosome assembly comes to a halt resulting
in accumulation of immature complexes in both the 30S and 50S assembly. Reconstitution
experiments with the 30S have been reported in the presence and absence of DnaK and in both
conditions a heat activation step was still required for complete assembly. The mechanism by
which DnaK facilitates ribosome assembly is undetermined although a direct affect on 30S
subunits has been suggested and debated [56-58]. These conflicting results regarding the role of
DnaK underscore the difficulty of correlating in vivo and in vitro ribosome assembly and
elucidating the precise function of assembly factors.

Bacterial GTPases implicated in Ribosome Assembly
GTPases occur in all domains of life and regulate key cellular processes [59,60]. The extent of
GTPase control of ribosome function is evident during translation where all steps, namely,
initiation, elongation and termination require GTPases [30,61-63]. In addition to regulating
ribosome function GTPases are also implicated in ribosome assembly. Nearly all bacterial

9

GTPases involved in ribosome assembly are essential for the cells and have been studied in
multiple bacterial species [22,23]. In addition these GTPases are conserved throughout evolution
and have eukaryotic homologs, several of which have also been implicated in eukaryotic
ribosome assembly in mitochondria and chloroplast [64,65]. Biochemical and genetic studies
have provided valuable functional insights into these GTPases [60,66]. However, their role in the
ribosome assembly process remains a mystery. Understanding the function of these GTPases in
ribosome assembly will uncover general principles of ribosome assembly in vivo and also
provide insights into how other multi-component complexes are regulated through GTPases.

GTPases implicated in the Assembly of the 50S subunit
RbgA
RbgA (ribosome biogenesis GTPase A) is an essential GTPase in B. subtilis that is required for
assembly of the 50S subunit. Cells depleted of RbgA are defective in ribosome assembly with a
drastic reduction in 70S ribosomes and an absence of the 50S subunit. Instead they accumulate a
complex that migrates at 45S and lacks three key r-proteins present in the mature 50S subunit
[67,68]. This is strikingly similar to the reconstitution intermediate RI50 seen in B.
stearothermophilus that is formed upon incubation of individual components at 30 °C and was
also found to be lacking in three ribosomal proteins indicating that the 45S complex seen in
RbgA depleted cells may be an in vivo intermediate in 50S assembly. The three r-proteins not
present in 45S are L16, L27 and L36 and all three proteins are located at key functional sites on
the mature 50S subunit [61]. R-proteins L16 and L27 are crucial to the formation of the A-site
and the P-site in the 50S subunit and are proposed to directly participate in the binding of the Asite tRNA and the P-site tRNA respectively [62,69]. E. coli deletion mutants of L27 and L36

10

show a severe growth defect while a deletion mutant of L16 has not been reported [69].
Assembly maps indicate that binding of L16 to the growing 50S subunit is a late step in the
assembly process [39].
RbgA can interact directly with the 45S complex and the mature 50S subunit though the latter is
strongest in the presence of a non-hydrolysable form of GTP [70,71]. Foot-printing assays have
indicated that RbgA protects nucleotides in the 23S rRNA near the A-site and the P-site [71].
These results together have suggested a model in which RbgA recruits r-proteins L16, L27 and
L36 to the 45S complex at a late stage in 50S assembly. The mechanism by which RbgA
participates in assembly of the 50S subunit is unknown. RbgA could either directly recruit one or
more of the r-proteins to the 45S complex or it could alter the conformation of the intermediate
leading to the incorporation of the r-proteins. Another key unanswered question is whether this
45S complex that is formed in the absence of RbgA is a true intermediate that can be matured to
a 50S subunit or is this a dead end complex that forms in absence of functional RbgA.
RbgA is a widely conserved protein and eukaryotic homologs of RbgA have been implicated in
ribosome assembly in chloroplast and mitochondria. Three homologs of RbgA in yeast Nog2,
Nug1 and Lsg1 are involved in maturation of 60S, the large ribosomal subunit in eukaryotes
[72,73]. Interestingly, Lsg1 is implicated to play a role in loading of r-protein Rpl10, the
eukaryotic homolog of L16, onto the pre-60S complex indicating that this may be an
evolutionarily conserved role of the RbgA protein family [74].
The eukaryotic protein that bears the most similarity to RbgA is the human Mtg1 protein that is
implicated in ribosome assembly in the mitochondria [75]. Mutations of mtg1 in yeast are
defective in mitochondrial protein synthesis leading to defective respiratory competence and
suppressors of mtg1 mutations map to the rRNA in large ribosomal subunit. Interestingly, the

11

human homolog of Mtg1 can partially rescue the respiratory deficiency of mutant mtg1 in yeast
indicating the conserved function of this protein across different organisms.
B. subtilis is an ideal system for investigating the function of RbgA in order to gain insight into
the role of essential GTPases in ribosome assembly. In eukaryotic systems defects in ribosome
assembly usually result in degradation of the intermediates or dead end complexes making it
difficult to elucidate the pathway of ribosome assembly. The accumulation of a stable 45S
complex in B. subtilis upon depletion of RbgA offers a unique opportunity to study ribosome
assembly in vivo and tease apart the role of RbgA in this process. This would also further our
understanding of the function of RbgA homologs in eukaryotic ribosome assembly.

ObgE
ObgE is an essential GTPase that is conserved from bacteria to humans and is involved in several
processes including ribosome assembly, modulation of the general stress response, sporulation
and chromosomal segregation and replication [22]. Several studies show that ObgE is involved
in assembly of the 50S subunit and ribosome function. Mutation or depletion of ObgE results in
a reduction of 70S ribosomes and an accumulation of individual subunits 50S and 30S [76]. The
50S subunits that accumulate in these strains are unstable and under conditions of high ionic
2+

strength and low Mg

concentration they dissociate into a 40S complex. Proteomic analysis of

this subunit indicated reduced amounts of L16, L33 and L34 and increased amounts of two
assembly factors RrmJ, a rRNA methyltransferase and RluC, a pseudouridine synthase [77]. In
addition overexpression of ObgE suppressed a temperature sensitive mutation in rrmJ. ObgE has
also been shown to specifically interact with r-protein L13. Recent evidence also indicates that
ObgE also interacts with SpoT, a bifunctional protein that both synthesizes and degrades the
12

second messenger (p)ppGpp [76]. These results suggest that ObgE could provide a link between
ribosome assembly and the synthesis of new rRNA and r-proteins.

YsxC
YsxC is a universally conserved protein that is essential for growth. Studies in E. coli indicate
that YsxC plays a role in cell division and the progression of the cell cycle. Subsequently studies
in B. subtilis showed that YsxC is involved in the assembly of the large ribosomal subunit [68].
Cells depleted of YsxC are defective in assembly of the 50S subunit and accumulate a complex
similar to RbgA depleted cells that migrates slightly slower at 44.5S and lacks three r-proteins
L16, L27 and L36. YsxC has also been shown to specifically bind to the 50S subunit in a GTP
dependant manner and can interact with r-proteins L1, L10 and L7/L12 [78]. Studies from
Staphylococcus aureus show that YsxC copurifies with the 50S subunit. The crystal structure of
YsxC from B. subtilis shows a patch of basic residues on the protein in the GTP-bound form but
not in the GDP bound form and this region could potentially be involved in interaction with the
ribosome [79]. However, the precise mechanism of interaction of YsxC with the ribosomes has
not been determined.

YphC
YphC is another widely conserved GTPase that is essential for growth and implicated in the
assembly of 50S subunits [68]. It is a unique protein which contains two GTP binding domains.
Studies on YphC from both E. coli as well as B. subtilis show that depletion of this protein
results in the reduction of 70S ribosomes and accumulation of individual subunits indicating
disruption of ribosome assembly and maturation [22]. However, the products of ribosome

13

assembly differ in each case. In B. subtilis YphC depletion leads to the accumulation of a
complex that migrates at 45S and also lacks proteins L16, L27 and L36 [68]. In contrast,
depletion in E. coli results in accumulation of the 50S subunit that are unstable compared with
mature 50S subunits seen in wild type cells [80]. Incubation of these 50S subunits in reduced
2+

levels of Mg

leads them to dissociate and result in a 40S complex that lacks r-proteins L9 and

L18. The differences between the E. coli and B. subtilis phenotypes further exemplifies the
importance of studying ribosome assembly in different bacterial systems.
Structural analysis of YphC provided further insight into guanine nucleotide control of the
interaction of the protein with the ribosome. The structure of YphC shows the presence of a KHdomain (a RNA binding domain) between the two GTP binding domains [81]. When the Nterminal domain is bound to GTP the KH domain is solvent exposed. However, when both G
domains are bound to GDP the KH domain is buried. This raises intriguing possibilities of
guanine nucleotide control of RNA binding to YphC. This is further supported by evidence that
GTP is required for association of YphC with the ribosome.

Era
Era is an essential GTPase shown play a role in assembly of 30S subunits and is known to bind
to 16S rRNA [82]. The protein binds directly to 30S subunits and crystal structures of Era with
the 30S subunit provide further insight into RNA-protein interaction. Era binds to the 30S
subunit at the interface that interacts with 50S subunit [82]. Mutations in Era result in reduction
of 70S subunits and accumulation of individual subunits. Together these results suggest that Era
plays a role in the association of the 30S subunit to the 50S subunit. However the precise role of
Era in maturation of the 30S subunit is unknown.
14

The immature 30S subunits that accumulate in Era mutants contain unprocessed 17S rRNA
instead of processed 16S rRNA which has led to a model that suggests that Era is involved in
processing of rRNA. However the appearance of 17S rRNA in 30S subunits is not limited to Era
mutants and has been observed in strains with mutations in genes that play a role in maturation
of the large subunit or inhibit translation.

RsgA
RsgA is implicated in the assembly of the 30S subunit and association of 30S and 50S subunits
[83]. However RsgA is different from other GTPases in that it is not essential for cell growth.
Studies from E. coli and B. subtilis show that RsgA interacts with the 30S subunit directly and
null mutants show a reduction in 70S ribosomes though the 30S and 50S subunits appear normal.
RsgA interacts with the 30S subunit close to the A-site and the P-site via its N-terminal domain
and deletion mutants of this domain cannot interact with the ribosome. The GTPase activity of
RsgA is stimulated ~160 fold upon interaction with the ribosome. Mutations in RsgA can be
suppressed by overexpression of Era and initiation factor IF-2.

YqeH
YqeH is found in a diverse group of bacteria and plants [84]. Depletion of YqeH causes a
complete loss of 30S subunits and a reduction in levels of 16S rRNA. Studies show that
premature 16S rRNA and breakdown products of 16S rRNA are seen indicating small subunit
assembly defects [85]. However there is a complete lack of immature 30S subunits making it
difficult to assess the function of the protein. The intrinsic GTPase activity of YqeH is not
enhanced by the 30S as seen in Era and RsgA [86]. Unlike other GTPases YqeH does not

15

directly interact with the ribosome. The YqeH homolog in Arabidopsis thaliana is involved in the
assembly of mitochondrial ribosomes as well as chloroplast ribosomes. The crystal structure of
YqeH from B. stearothermophilus indicates that C-terminal domain of the protein interacts with
rRNA.

Unique features of Ribosome Assembly GTPases
All GTPases are believed to have evolved from a common ancestor and this is reflected in the
conservation of the three dimensional fold as well as the sequence motifs of the G-domain. Five
conserved motifs G1-G5 form the G-domain that is responsible for binding and hydrolysis of
GTP. The G1 motif or P-loop is characterized by GXXXXGKS/T and interacts with the β and γ
phosphates of GTP. The G2 motif is characterized by a single conserved T residue that
2+

coordinates the Mg

2+

coordinates the Mg

ion. The G3 motif, characterized by DXXG of which residue D also
ion and residue G interacts with the γ phosphate of GTP. Motifs G2 and

G3 are also called switch I and switch II and acquire distinct conformations in GTP and GDP
bound forms. The G4 motif, characterized by NKXD is responsible for specificity of binding to
guanine nucleotide. The G5 motif, characterized by SAK/L is not universally present in all
GTPases. Although all GTPases share these conserved structural features there are some
characteristics that are specific to ribosome assembly GTPases.
The GTPase activity of these enzymes can be stimulated by a GTPase activating protein (GAP)
that is thought to provide the arginine residue in order to neutralize the negative charge buildup
on the β-and γ-phosphate. Hence, GAPs stabilize the transition state of the GTPase reaction [87].
In several ribosome assembly GTPases the ribosomal subunits stimulates GTPase activity
suggesting that a rRNA residue(s) could also act as a GAP.
16

HAS-GTPases
All GTPases that are implicated in ribosome assembly are members of the HAS (hydrophobic
amino acid substituted) family of GTPases [88]. In classical GTPases the catalytic residue Q
follows the G3 motif. This residue activates the nucleophilic water molecule to attack the γphosphate bond. In HAS-GTPases a hydrophobic amino acid is found in place of this catalytic Q
and thus the mechanism of GTP hydrolysis in ribosome assembly GTPases is not known. It has
been proposed that the catalytic residue could be provided in cis by another part of the protein or
in trans by another factor. The structure of RbgA shows that the isoleucine residue found in place
of the catalytic glutamine is retracted from the active site which leaves a void for a residue from
another protein or factor or an rRNA residue could facilitate GTP hydrolysis.

Circularly permuted GTPases
Some ribosome assembly GTPases such as RbgA, RsgA and YqeH are part of the circularly
permuted GTPase family (cpGTPases) [89]. In this family the G4 motif preceeds the G1 motif
resulting in a unique circular permutation of the G-domain. While there are subtle structural
differences between cpGTPases and classical GTPases the structure of the GTP binding pocket
remains relatively unaltered. All of the cpGTPases for which a function has been described or
predicted either interact with RNA or are involved in ribosome assembly.
Another feature of these ribosome assembly cpGTPases is that the G3 motif (switch II) is no
longer anchored to the G4 motif. Instead switch II is linked to a C-terminal domain which is
necessary to stabilize the switch II region. This arrangement in ribosome assembly GTPases
would allow for guanine nucleotide occupancy to control the conformation and/or movement of
the C-terminal domain of the protein. How this circular permutation of the G-domain allows the

17

proteins to interact with the ribosome remains unanswered. Another relevant question in this
context concerns the role of the C-terminal domain in GTPase function in these enzymes.

Affinity for guanine nucleotides
Classical GTPases of the Ras superfamily bind guanine nucleotides in the nanomolar to
picomolar range. Such a high affinity for GTP and GDP indicates that guanine nucleotide
exchange factors (GEFs) are necessary for catalytic turnover. However this is not the case for the
ribosome assembly GTPases that have been characterized so far. The ribosome assembly
GTPases bind to guanine nucleotide in the micromolar range approximating the physiological
guanine nucleotide concentration in the cells during stress conditions with RbgA and RsgA
displaying the weakest binding affinities. Further, studies have shown that both Era and ObgE
have fast nucleotide exchange rates. Together, these data suggest that ribosome assembly
GTPases may not require GEFs and are likely regulated directly by the GTP/GDP ratio in the
cells. Such a system would allow ribosome assembly to be directly coupled to the energy status
of the cell. Further biochemical characterization of ribosome assembly GTPases is required in
light of these observations.

Possible mechanisms by which GTPases could participate in Ribosome Assembly
There is much evidence of the crucial role of GTPases in ribosome assembly. However the
mechanisms by which these GTPases regulate ribosome assembly remains a mystery. Two
possible mechanisms have been suggested based on the available data.
Ribosome GTPases could play a role in directly recruiting r-proteins or other assembly factors to
the ribosome during assembly. Such a function has been shown for yeast GTPase Bms1 which

18

directly recruits assembly factor Rcl1 to the pre-40S complex. Rcl1 is an essential enzyme
required for processing of pre-rRNA during ribosome maturation. This was the first
demonstration of direct recruitment of an assembly? factor by ribosome assembly GTPase. To
date no such function has been demonstrated in bacterial GTPases.
It is possible that ribosome assembly GTPases could regulate the activity of RNA helicases and
thus control rRNA structure. In the regulation of spliceosome in yeast GTPase Snu114p
stimulates the RNA helicase activity of DEAD box helicase Brr2p and thus controls the
assembly and disassembly of the spliceosome. A similar interplay between ribosome assembly
GTPases and RNA helicases could be possible during the assembly process.

Research over the past decade has shown that GTPases play a crucial role in assembly of
bacterial, mitochondrial and chloroplast ribosomes. The fact that GTPases are required for
ribosome assembly in all three kingdoms of life underscores their critical importance for the
biogenesis process. What remains to be addressed is how these GTPases participate in ribosome
assembly on a molecular level.

19

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20

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79. Ruzheinikov, S.N., et al. (2004) Analysis of the open and closed conformations of the GTPbinding protein YsxC from Bacillus subtilis. J Mol Biol 339: 265-78.
80. Bharat, A., Jiang, M., Sullivan, S.M., Maddock, J.R., Brown, E.D. (2006) Cooperative and
critical roles for both G domains in the GTPase activity and cellular function of ribosomeassociated Escherichia coli EngA. J Bacteriol 188: 7992-6.
81. Muench, S.P., Xu, L., Sedelnikova, S.E., Rice, D.W. (2006) The essential GTPase YphC
displays a major domain rearrangement associated with nucleotide binding. Proc Natl Acad Sci
U S A 103: 12359-64.
82. Sharma, M.R., et al. (2005) Interaction of Era with the 30S ribosomal subunit implications
for 30S subunit assembly. Mol Cell 18: 319-29.
83. Goto, S., Kato, S., Kimura, T., Muto, A., Himeno, H. (2011) RsgA releases RbfA from 30S
ribosome during a late stage of ribosome biosynthesis. EMBO J 30: 104-14.
84. Uicker, W.C., Schaefer, L., Koenigsknecht, M., Britton, R.A. (2007) The essential GTPase
YqeH is required for proper ribosome assembly in Bacillus subtilis. J Bacteriol 189: 2926-9.
85. Anand, B., Surana, P., Bhogaraju, S., Pahari, S., Prakash, B. (2009) Circularly permuted
GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly.
Biochem Biophys Res Commun 386: 602-6.
86. Anand, B., Surana, P., Prakash, B. (2010) Deciphering the catalytic machinery in 30S
ribosome assembly GTPase YqeH. PLoS One 5: e9944.
87. Scheffzek, K., Ahmadian, M.R. (2005) GTPase activating proteins: structural and functional
insights 18 years after discovery. Cell Mol Life Sci 62: 3014-38.

26

88. Mishra, R., Gara, S.K., Mishra, S., Prakash, B. (2005) Analysis of GTPases carrying
hydrophobic amino acid substitutions in lieu of the catalytic glutamine: implications for GTP
hydrolysis. Proteins 59: 332-8.
89. Anand, B., Verma, S.K., Prakash, B. (2006) Structural stabilization of GTP-binding domains
in circularly permuted GTPases: implications for RNA binding. Nucleic Acids Res 34: 2196-205.

27

CHAPTER 2
Biochemical characterization of the ribosome assembly GTPase RbgA in Bacillus subtilis

The contents of this chapter were published in Achila D., Gulati M., Jain N. and Britton
R.A. (2012) Biochemical characterization of ribosome assembly GTPase RbgA in Bacillus
subtilis. J Biol Chem. 287:8417-8423. MG contributed to Table 2.3 and Figure 2.5. MG
and NJ collaborated on Figure 2.3 and DA contributed to Figures 2.1, 2.2 and 2.4.

28

Abstract
The ribosome biogenesis GTPase A protein (RbgA) is involved in the assembly of the large
ribosomal subunit in Bacillus subtilis and homologs of RbgA are implicated in the biogenesis of
mitochondrial, chloroplast, and cytoplasmic ribosomes in archaea and eukaryotes. The precise
function of how RbgA contributes to ribosome assembly is not understood. Defects in RbgA
give rise to a large ribosomal subunit that is immature and migrates at 45S in sucrose density
gradients. Here we report a detailed biochemical analysis of RbgA and its interaction with the
-1

ribosome. We found that RbgA, like most other GTPases, exhibits a very slow kcat (14 h ) and
has a high Km (90 µM). Homology modeling of the RbgA switch I region using the K-loop
+

GTPase MnmE as a template suggested that RbgA requires K ions for GTPase activity, which
was confirmed experimentally. Interaction with 50S subunits, but not 45S intermediates,
increases GTPase activity ~55 fold. Stable association with 50S subunits and 45S intermediates
was nucleotide dependent and GDP did not support strong interaction with either of the subunits.
GTP and GMPPNP were sufficient to promote association with the 45S intermediate while only
GMPPNP was able to support binding to the 50S subunit, presumably due to the stimulation of
GTP hydrolysis. These results support a model in which RbgA promotes a late step in ribosome
biogenesis and that one role of GTP hydrolysis is to stimulate dissociation of RbgA from the
ribosome.

29

Introduction
Ribosome assembly is a complex, tightly regulated process that involves the coordinated
assembly of 3 RNA molecules and over 50 proteins [1,2]. Although more than 150 accessory
proteins required for the assembly of ribosomes in eukaryotes have been identified [3], relatively
few proteins dedicated to the assembly of ribosomes have been discovered in bacteria [4-6].
Several ribosome associated GTPases (RA-GTPase) such as RbgA (YlqF), Era, YqeH, YphC
(EngA), CgtEA, YloQ (YjeQ, RsgA) and YsxC have been implicated in the assembly of either
the 50S or 30S ribosomal subunits in bacteria, however their precise functions in ribosome
assembly has remained elusive [6-13]. The RA-GTPase RbgA is required for a late maturation
step of the 50S subunit in B. subtilis [8,10] and its homologs have been shown to be required for
the assembly of the large ribosomal subunit in eukaryotes [14-17].
Depletion of RbgA in B. subtilis stalls 50S subunit assembly resulting in the accumulation of a
large ribosomal intermediate that migrates more slowly (45S) through a sucrose gradient and
lacks three ribosomal proteins, L16, L27, and L36 [8,10,11]. RbgA can interact with the 45S
intermediate and 50S subunits, but the latter interaction was only observed in the presence of a
non-hydrolyzable analog of GTP [8,10,11]. It was also observed that intact 50S stimulates the
GTPase activity of RbgA [8]. These observations led to proposal of a model in which RbgA
promotes a late step in ribosome assembly and that GTPase activation of RbgA occurs upon
correct assembly of the 50S subunit followed by dissociation of RbgA from the subunit [8,10]. In
this way RbgA would serve as a checkpoint to ensure proper formation of the 50S subunit.
However, it was later reported that the GTPase activity of RbgA is maximally stimulated by the
45S intermediate (referred to as pre50S in this model) and “free” 50S subunits but not by mature
50S subunits (those isolated by dissociation of subunits from 70S ribosomes) [18]. These

30

additional results led to a revised model in which RbgA promotes a GTPase dependent structural
rearrangement of the 45S complex that then allows L16 and L27 to subsequently bind. GDP
bound RbgA remains associated with the ribosome until some undetermined signal triggers the
release of RbgA [18]. The later model posits that structural difference exists between “free” 50S
and mature 50S subunits to explain the differential activation of RbgA by free 50S subunits.
In order to address these two conflicting models we undertook a more thorough biochemical
analysis of RbgA and explored its interaction with the ribosome in details. Our results
demonstrate that the GTPase activity of RbgA is maximally stimulated by both mature and free
50S subunits, more than ten times over the stimulation observed with the 45S intermediate.
Interaction assays of RbgA with the different ribosomal subunits shows that GDP-bound RbgA
does not stably associate with the ribosome and suggests that the GTPase activity of RbgA
promotes dissociation from the ribosome. We discuss our findings in the context of the two
conflicting models and further clarify the role of GTPase activity in RbgA function during
ribosome assembly.

Materials and Methods
Growth conditions and strain construction
All experiments were performed at 37°C in Luria-Bertani (LB) medium. When necessary,
antibiotics were added at the following concentrations: Chloramphenicol (5 μg/ml), Ampicillin
(100 μg/ml). All B. subtilis strains used in this study were derived from the wild-type strain
JH642 (RB247). B. subtilis RB301 (Pspank-rbgA) and RB418 (Pspank-infB) strains were
constructed as previously described [10]. RB301 and RB418 accumulate 45S and 50S subunits,
respectively, when grown in the absence of IPTG and were used to purify 45S and free 50S

31

ribosomal subunits. E. coli BL21(DE3) transformed with plasmids containing full-length rbgA
placed under the control of the T7 promoter was used to overexpress RbgA proteins [10].

Purification of RbgA proteins
RbgA protein with histidine tag (His6) at the C-terminus was isolated as previously described
[10]. Briefly, E. coli BL21(DE3) cells transformed with plasmid containing full-length rbgA
under IPTG inducible T7 promoter [10] were grown to OD600 of 0.5 at 37°C in LB medium
containing 100 μg/mL ampicillin then induced by addition of 1 mM isopropyl β-thiogalactoside
(IPTG). The cells were harvested by centrifugation after 3 hours and resuspended in binding
buffer (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl and 20 mM imidazole). The cells were
lysed by three consecutive passes through a French press at between 1400 to 1600 psi then
clarified by centrifugation at 16000xg for 20 min. RbgA-His6 was isolated from the cell lysate
by affinity chromatography using HisTrapHP (Ni-NTA resin column) from GE Healthcare
BioScience on a BioRad BioLogic LP chromatography system. Cell lysate was injected into the
column pre-equilibrated with binding buffer and allowed to equilibrate for 5 minutes then
washed with 5 column volumes of wash buffer (20 mM sodium phosphate, pH 7.5, 0.5 M NaCl
and 60 mM imidazole) followed by step elution with binding buffer containing 250 mM
imidazole. Elution was monitored by UV absorption at 280 nm and RbgA containing fractions
were verified by SDS-PAGE gel then concentrated and desalted by exchanging buffer to
desalting buffer (50 mM Tris-HCl pH 7.5, 750 mM KCl, 5 mM MgCl2, 20 mM imidazole, 2 mM
DTT and 10% glycerol) and then to storage buffer (50 mM Tris-HCl, pH 7.5, 750 mM KCl, 5
mM MgCl2, 2 mM DTT and 10% glycerol). Purity of isolated concentrated RbgA was verified

32

by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) before storage at -20ºC. K59A, P129R,
S134A and F180A mutations were introduced into rbgA by the QuikChange II XL kit
(Stratagene).

Preparation of ribosomal particles
Mature 50S, free 50S and 45S large ribosomal subunits were prepared by sucrose density
centrifugation [19,20]. 50S and 45S complexes were isolated from lysates of RB418 and RB301
cells respectively. Mature 50S subunits were isolated by subjecting the RB247 lysate to low
2+

Mg

2+

buffer (buffer B containing 1 mM Mg ), thereby dissociating the 70S ribosome into 50S

and 30S subunits. The cells were grown were grown to OD600 of 0.5 at 37°C in LB medium with
or without IPTG. RB301 and RB418 cells were grown without IPTG for several generations to
deplete the cells of RbgA and IF-2, respectively, until a doubling time of about 150 min was
reached. Chloramphenicol (Sigma) was added to a final concentration of 100 μg/ml 5 minutes
prior to harvesting. Cells were harvested by centrifugation at 5000xg for 10 min and resuspended
in lysis buffer (10 mM Tris-HCl (pH 7.5), 60 mM KCl, 10 mM MgCl2, 0.5% Tween 20, 1 mM
DTT, 1× Complete EDTA-free protease inhibitors (Roche) and 10 U/ml RNase-free DNase
(Roche). Cells were lysed by three consecutive passes through a French press set at 1400 to 1600
psi, then clarified by centrifugation at 16000xg for 20 minutes. Clarified cell lysates were loaded
on top of 10-25% sucrose density gradients equilibrated in buffer B (10 mM Tris-HCl, pH 7.6,
10 mM MgCl2, 50 mM NH4Cl) and centrifuged using an SureSpin 630 rotor (Sorvall) for 4.5
hours at 30,000 rpm. Gradients were then fractionated on BioRad BioLogic LP chromatography
system by monitoring UV absorbance at 254 nm. Fractions corresponding to ribosomal subunits

33

of interest were pooled, concentrated using 100 kDa cutoff filters (Millipore) and stored in buffer
A (10 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 60 mM KCl and 1 mM DTT) at -80°C. To purify
mature 50S subunits, RB247 cells were grown in LB medium at 37°C to an OD600 of 0.5,
2+

harvested, and resuspended in lysis buffer containing only 1 mM Mg

concentration. The lysate

was incubated on ice for 30 minutes and then centrifuged for 14 hours at 20,600 rpm through a
2+

25-40% sucrose gradient prepared in buffer B containing 1 mM Mg

(10 mM Tris-HCl, pH 7.6,

1 mM MgCl2, 50 mM NH4Cl). 50S subunit peak fractions were pooled, concentrated, and stored
in buffer A.

Characterization of GTPase activity of RbgA
GTP hydrolysis activity of RbgA was determined incubating RbgA with GTP for 30 minutes
then measuring the released free phosphate by the malachite green–ammonium molybdate
colorimetric assay (BioAssays) [21]. Intrinsic GTPase activity was assayed in triplicate at a
constant RbgA protein concentration of 2 µM and a range of GTP concentrations (8 to 650 μM)
(USB corp.). GTPase reactions was started by addition of protein to GTP solutions at 37°C in
reaction buffer (50 mM Tris-HCl, pH 7.5, 200 mM KCl, 5 mM MgCl2, and 1 mM DTT) for 30
minutes then terminated by addition of Malachite reagent. We determined that under these
conditions GTP hydrolysis was in the linear range of the assay and less than 10% of the substrate
had been consumed. Released phosphate was detected by monitoring color formation at 620 nm
using a 96-well plate reader (Tecan Sunrise). Experiments were repeated a minimum of three
times and values for Km and kcat were calculated by fitting the data to the Michaelis-Menten

34

equation with nonlinear regression curve fitting using GraphPad Prism (Graph-Pad Software
Inc.; version 5.0).

Stimulation of GTPase activity of RbgA by ribosomal subunits
To probe the effect of ribosomal subunits on the GTPase activity of RbgA, 100 nM of purified
ribosomal subunits (mature 50S subunits from dissociated 70S, free 50S subunits from IF-2
depleted cells, and 45S subunits from RbgA depleted cells) were individually incubated with 100
nM RbgA for 15 min at 37°C in reaction buffer (50 mM Tris-HCl, pH 7.5, 200 mM KCl, 5 mM
MgCl2, and 1 mM DTT) then added to 8-650 µM GTP to start the reaction. We predetermined
that under these conditions the values were in the linear range of the assay. The activity of
RbgA-subunit complex was determined by standard malachite green assay as described above
for RbgA proteins alone. Three biological replicates (independent RbgA and ribosome
preparations) were performed, each with three technical replicates.

Importance of potassium ion for GTPase activity of RbgA
+

For testing with the GTPase activity in the presence of K , the protein was purified as described
above and stored in storage buffer (50 mM Tris-HCl, pH 7.5, 750 mM KCl, 5 mM MgCl2, 2 mM
+

DTT,10% glycerol).To test the effects of Na , the protein was exchanged and stored in NaCl
buffer (50 mM Tris-HCl, pH 7.5, 750 mM NaCl, 5 mM MgCl2, 2 mM DTT and 10% glycerol).
The intrinsic activity of RbgA was assayed by incubation of 2 µM of RbgA with 200 µM GTP
for 15 min at 37°C. Stimulation of GTPase activity was tested by incubation of 100 nM of free

35

50S subunits with 100 nM RbgA and 200 µM GTP for 15 min at 37°C. The activity was
determined by standard malachite green assay as described above.

Homology modeling of the K-loop of RbgA
Homology modeling of the K-loop of RbgA was carried out with Modeler [22] using MnmE as a
template. A multiple sequence alignment in which K-loop of MnmE was used as a template to
model the RbgA K-loop, which is disordered in available crystal structures, while for the
remainder of RbgA the existing crystal structure was used (PDB: 1PUJ).A total of 20 initial
models were made the best model was chosen based on the lowest energy (molpdf) and the
lowest discrete objective protein energy (DOPE) function. A ligand based superimposition of
RbgA (PDB: 1PUJ) and MnmE (PDB: 2GJ8) was carried out using ligalign script [23] in
PyMOL [24]. The figure was generated by using Chimera [25].

Interaction between RbgA and ribosomal particles
RbgA protein and ribosomal particles were purified as described above. The RbgA subunit
binding assay was performed as previously described [8]. The protocol was however modified to
include 15 min incubation with a high salt buffer, buffer C (10 mM Tris-HCl pH 7.5, 60 mM
KCl, 500 mM NH4Cl, 10 mM MgCl2 and 1 mM DTT) to further test the specificity of
RbgA/subunits interaction. Briefly, 60 pmol of RbgA was pre-incubated with 1.5 mM of
different guanine nucleotides for 15 min followed by addition of 10 pmol purified subunits. The
binding was allowed to proceed for 15 min at 37°C, then centrifuged once through Microcon 100
filters (Millipore) with a cut-off of 100 kDa. The RbgA-subunit complexes were washed once
with buffer A, twice with buffer C before elution. Ribosome bound RbgA was detected by

36

separating the complexes on NuPAGE 12% Bis-Tris SDS gels (Invitrogen) followed by Western
blot analysis, using rabbit polyclonal anti-RbgA and HRP-conjugated goat anti-rabbit antibodies.
RbgA

specific

bands

were

visualized

with

the

Western

Lightning

(PerkinElmer)

chemiluminescent detection system.

Results
RbgA has a low intrinsic GTPase activity
Most RA-GTPases have low intrinsic GTPase activity in the absence of an effector protein or
molecule. To characterize the intrinsic GTPase activity of RbgA, we purified RbgA-His6 and
measured GTP hydrolysis using a malachite green assay. Previously we have shown that the Cterminal fusion of six histidine residues to RbgA yields a functional protein that can support
wild-type growth of B. subtilis as well as interaction with the ribosome [10]. We performed a
steady state kinetic analysis of RbgA GTPase activity using the RbgA-His6 protein (Figure 2.1).
-1

-1 -1

Wild-type RbgA had a kcat of 14 h , a Km of 90 µM, and a kcat/Km of 47 M s (Table 2.1). To
confirm that the low rate of hydrolysis observed was due to RbgA and not a contaminant, we
created three mutants that altered important residues in the P-loop (P129R, S134A) or the G4
region that specifies binding for guanine nucleotides (K59A) and tested their ability to hydrolyze
GTP (Figure 2.1 and Table 2.2). All three mutants displayed lower GTPase activity and a
reduced kcat/Km, indicating that the GTPase activity observed for wild-type RbgA was not due
to a contaminant.

37

The GTPase activity of RbgA is maximally stimulated by mature or free 50S ribosomal
subunits
To better understand the role of GTPase activity in the function of RbgA, we characterized GTP
hydrolysis of RbgA in the presence of 50S subunits and 45S intermediates. We tested three types
of large ribosomal subunits: mature 50S subunits isolated by dissociating subunits from 70S
ribosomes, free 50S subunits isolated by depleting initiation factor 2 (IF-2) and 45S
intermediates purified from RbgA depleted cells. Previous work had indicated that 45S
intermediates and free 50S subunits had higher stimulatory activity than mature 50S subunits
[18]. Ribosomal subunits were mixed with RbgA in a ratio of 1:1 and the GTPase activity of
RbgA was monitored using a malachite green assay. Both mature and free 50S subunits were
capable of stimulating the GTPase activity of RbgA ~55-fold (Figure 2.2 and Table 2.1). In
contrast the 45S intermediate isolated from RbgA-depleted cells showed a markedly reduced
ability to stimulate the GTPase activity of RbgA (5-fold) over intrinsic GTPase activity.
Michaelis-Menten analysis of the GTPase activity of RbgA indicated that the increase in GTPase
activity with 50S subunits was primarily governed by an increase in kcat (Table 2.3). Conversely,
interaction with the 45S intermediate decreased the Km of RbgA for GTP to 4 µM, indicating a
tighter affinity for GTP in the presence of the 45S complex while kcat was largely unaffected.
Our results show that GTPase activity of RbgA is maximally stimulated by the 50S subunits
(free and mature), in contrast to previously published results [18].

38

Potassium is required for optimal GTPase activity
Recent work has indicated that three translation associated GTPases (MnmE, FeoB and YqeH)
+

utilize a unique mechanism for GTP hydrolysis in which a K ion participates in the activation of
+

GTP hydrolysis [26-28]. The structures of these GTPases demonstrate that a K ion functions as
a GTPase Activating Element (GAE), analogous to GTPase activating proteins (GAP) in
eukaryotic GTPases.
+

This K ion occupies a position, usually occupied by an arginine residue, referred as “arginine
+

finger” in GAPs of Ras related GTPases [29]. The positive charge provided by arginine or K at
this position stabilizes the transition state by neutralizing negative charges that builds up during
+

+

the transition state. In GTPases that require K , the K ion is held in position by a region of the
GTP binding domain that has been designated the “K-loop”[28]. The K-loop is located upstream
+

of switch I and interacts with K through the peptide backbone of amino acids and shields the
+

K ion from the bulk solvent. An additional side-chain interaction is provided by a conserved
asparagine residue situated within the P-loop. Inspection of the primary sequence of RbgA
indicated a possible K-loop type sequence; however this part of the protein is not resolved in any
of the crystal structures of RbgA to date. Therefore, we modeled this K-loop in RbgA using
transition state structure of MnmE [28] (Figure 2.3A). Out of 20 homology models generated,
the most energetically favorable model shows a high degree of similarity to the K-loop of
MnmE. Moreover, the asparagine residue from P loop (GIPNVGKS in RbgA), that makes a
+

direct contact with the K in other 16 activated GTPases, is also oriented similar to P loop

39

asparagine in the RbgA crystal structure (Figure 2.3B). This suggests that RbgA may also utilize
+

K in the activation of GTPase activity similar to MnmE, FeoB and YqeH.
+

Next, we investigated whether K was required for maximal stimulation of RbgA GTPase
activity. The intrinsic GTPase activity and stimulation of RbgA GTPase was monitored in the
presence of 250 mM NaCl or KCl. The intrinsic GTPase activity of RbgA is reduced 133-fold
+

when NaCl is substituted for KCl, demonstrating that K is required for optimal RbgA GTPase
activity (Table 2.3). The stimulation of RbgA GTPase activity by the ribosome is also affected
+

by Na but to a lesser degree, showing a 22-fold reduction in GTPase activity when co-incubated
+

+

with 50S subunits compared to K conditions. These results show that K

is required for

maximal GTPase activity.

RbgA interacts with the 50S ribosomes and 45S intermediates in a nucleotide specific
manner
Previous work showed that RbgA interacts with the large ribosomal subunit when incubated with
a non-hydrolyzable analog of GTP [8]. In order to explore interaction of RbgA with the subunits
in detail, the interaction of RbgA with both purified 50S subunits and 45S intermediate was
characterized in the presence of GTP, GDP, and GMPPNP. Saturating levels of guanine
nucleotides were used to ensure all RbgA molecules were bound to nucleotide. 45S intermediates
or 50S subunits were incubated with RbgA for 15 minutes at 37°C and were spun through a 100
kDa cutoff microcon filter to remove unbound RbgA. RbgA bound to the subunits was retained
on top of the filter and was detected using RbgA specific polyclonal antibodies. We found that
RbgA associates most stably with the 45S subunit in the GTP and GMPPNP bound forms and
40

that binding is greatly reduced in the presence of GDP (Figure 2.4). In the presence of the 50S
subunit, RbgA binding is maximally enhanced in the presence of GMPPNP but is greatly
reduced in the presence of GTP and GDP. The difference between GTP and GMPPNP is likely
due to GTP hydrolysis in the presence of the 50S ribosome, resulting in dissociation from the
ribosome. We also tested if the alarmone, pppGpp, affected RbgA interaction with the ribosome.
We found that addition of pppGpp enhanced the interaction of RbgA with both the 45S
intermediate and 50S subunits.

Discussion
Previous genetic and biochemical studies aimed at elucidating the role of RbgA in large
ribosomal subunit assembly have yielded two conflicting models. One model posits that RbgA
couples a final maturation step of the 50S subunit with the activation of GTP hydrolysis, which
signals RbgA to leave the ribosome [8,10]. This maturation step involves the incorporation of
ribosomal proteins L16, L27, and L36. However, a revised model was proposed suggesting that
RbgA uses GTP hydrolysis to induce a conformational change in the 45S subunit that is
independent of the incorporation of L16, L27, and L36 [18]. The revised model is based upon the
finding that the 45S subunit can maximally stimulate RbgA GTPase activity in vitro, although
one major complication of the model is that free 50S subunits, but not mature 50S subunits, can
also stimulate GTP hydrolysis to the same level as the 45S intermediate. Because both of these
models rely on limited biochemical evidence, we undertook a detailed analysis of RbgA
interaction with the ribosome to clarify the role of GTP hydrolysis in RbgA function.
Several lines of evidence presented here support the model in which RbgA utilizes GTPase
activity to dissociate from the ribosome. First, GTPase stimulation by 50S subunits, free or

41

mature, was ten times more than that observed with 45S intermediates. Second, RbgA interaction
with 50S subunits was nucleotide dependent, with reduced levels of binding observed in the
presence of GTP and GDP compared to the non-hydrolyzable analog GMPPNP. In contrast, the
45S intermediate displayed similar binding in the presence of GTP and GMPPNP with greatly
reduced binding in the presence of GDP. Decreased binding of RbgA to the 50S subunit in the
presence of GTP, but not GMPPNP, is consistent with GTP hydrolysis governing dissociation of
RbgA from the ribosome. Third, steady state kinetic analysis of RbgA in the presence of the
different ribosomal subunits showed that the kcat of RbgA was dramatically increased only in the
presence of the 50S subunit. This indicates that the 50S, but not the 45S intermediate, serves a
GAP-like function for RbgA. Additionally Km was decreased only in presence of 45S but not
50S, which suggest a cooperative binding of 45S and GTP to RbgA. These data support a model
in which the GTPase activity of RbgA is required to dissociate RbgA from the 50S subunit. It is
possible that RbgA either serves to monitor correct ribosome formation with its GTPase activity
or that it couples the hydrolysis of GTP to a conformational change in the subunit while
subsequently dissociating from the subunit.
An important question to address is why we observe maximal stimulation of RbgA GTPase
activity in the presence of 50S subunits (free or mature) while previous work shows maximal
stimulation with the 45S intermediate and free 50S subunits but not mature subunits [18]. Our
steady-state kinetic analysis of RbgA indicates a potential answer to this question. The Km for
RbgA in the absence of the ribosome is ~90 µM, and given the slow forward reaction in the
absence or presence of the ribosome the Km is a good estimate for the KD of RbgA for GTP. In
the presence of 50S subunits the Km is only marginally reduced (62 µM for mature 50S and 32
42

µM for free 50S subunits). However in the presence of the 45S intermediate the Km of RbgA is
reduced to 4 µM, indicating that interaction with the 45S intermediate fosters tighter association
with GTP. In previous work GTPase assays were performed using a concentration of 10 µM
GTP, well below the Km of RbgA for GTP in the presence of the 50S subunit [18]. These assay
conditions would result in most of the RbgA protein being unbound to GTP and should
underestimate the activity of RbgA in the presence of 50S subunits. Since GTP concentration in
the cell is in the range of 0.5-5 mM [30,31], our assay likely reflects a more physiologically
relevant condition. Finally, our results demonstrate that RbgA is a member of a growing family
+

of GTPases that utilize K as part of the mechanism of GTP hydrolysis. Previous work on the
+

biochemistry of RbgA was performed with NH4Cl [18]. While NH4 supports hydrolysis of GTP
+

+

in YqeH, it is reduced 2-fold in its activity indicating that NH4 is not sufficient to replace K

for full activity [26]. Future experiments on the biochemistry of RA-GTPases thus warrant
+

investigation of K as a possible GTPase activating element.
The results presented here support a model in which the guanine nucleotide state of RbgA
regulates the association of RbgA with the ribosome (Figure 2.5). GDP significantly inhibits
RbgA interaction with either the 45S intermediate or the 50S subunit, and the interaction with the
50S subunit is only stabilized in the presence of non-hydrolyzable analogs of GTP. These results
indicate that GTP hydrolysis promotes RbgA dissociation from the 50S subunit, similar to other
translation factor GTPases such as EF-Tu. EF-Tu couples proper accommodation of the aa-tRNA
to GTPase activity, resulting in release of EF-Tu from the ribosome [32]. We propose that RbgA
promotes a late step in large subunit assembly that allows the stable incorporation of ribosomal

43

proteins L16, L27, and L36. While the nature of this rearrangement is still unknown, once
properly achieved we propose that RbgA “senses” the correct assembly of the ribosome which in
turn promotes GTP hydrolysis and RbgA dissociation. Alternatively, RbgA could act directly on
the 45S intermediate independently of incorporation of L16, L27, or L36. Finally, we previously
proposed that regulating a late assembly process would allow RbgA to act as a checkpoint
governing the release of active 50S subunits into the translation active pool of ribosomes in the
cell. Previous work with BipA has also shown that pppGpp can regulate GTPase association with
ribosomal subunits [33]. It is intriguing that alarmone pppGpp enhances association of RbgA
with both 45S and 50S subunits, given the fact that RbgA likely binds to the subunit interface
and would block association with the 30S subunit. Under times of translational stress pppGpp
could block the additional maturation of 45S intermediates and prevent newly formed 50S
subunits from participating in translation.

44

Ribosome
none
50S free
50S mature
45S intermediate

-1

kcat (h )
14 ± 2
755 ± 207
807 ± 158
69 ± 16

Km (µM)
90 ± 13
32 ± 6
62 ± 16
4±3

-1 -1

kcat/Km(M s )
43
6533
3616
4791

Table 2.1 Kinetic parameters of RbgA in the presence and absence of ribosomal subunits.
The intrinsic activity of RbgA was assayed by incubation of 2 µM of RbgA with various
concentrations of GTP for 30 min at 37°C. Stimulation of RbgA GTPase activity by ribosomes
was assessed by determining the GTP hydrolysis rates of RbgA in the presence of various
purified ribosomal particles. 100 nM of purified ribosome was incubated with 100 nM RbgA for
15 min at 37°C in the presence of various concentrations of GTP and assayed as described in the
Materials and Methods section. Standard deviations of the mean of three biological replicates are
presented.

45

RbgA Proteins k (h-1)
cat
Wild Type
15 ± 0.4

Km (µM)

kcat/Km (M s )

-1 -1

72 ± 5

58

F180A
P129R

18 ± 0.8
8 ± 0.8

65 ± 9
290 ± 58

77
8

K59A

10 ± 1

286 ± 60

10

S134A

7 ± 0.6

146 ± 32

13

Table 2.2 Intrinsic GTPase activity of RbgA mutants.
The intrinsic activity of RbgA was assayed by incubating 2 µM of RbgA with various
concentrations of GTP for 30 min at 37°C. P-loop mutants (P129R and S134A), linker region
mutant (F180A) and G4 region mutant K59A were purified and assayed in a similar manner to
RbgA wild type. Released free phosphate was determined by Malachite green assay. The
experiments were done at least three times and the values of kcat and Km were determined by
fitting the Michaelis-Menten equation using nonlinear regression algorithms provided by the
GraphPad Prism software (GraphPad Software, Inc.). Standard deviations of the mean of at least
three biological replicates are shown. As expected, the P-loop and G4 mutations increase the Km
and lower the kcat. The linker region mutation (F180A) has no remarkable effect on the GTPase
activity of RbgA.

46

Intrinsic GTPase
activity

Stimulation of GTPase activity in
the presence of the 50S subunit

V = pmol phosphate/pmol RbgA/ min
RbgA 250 mM KCl
RbgA 250 mM NaCl

0.26 ± 0.02
< 0.003

13.8 ± 0.85
0.78 ± 0.1
+

+

Table 2.3 GTPase activity of RbgA in the presence of Na and K ions.
The intrinsic activity of RbgA was assayed by incubation of 2 µM of RbgA with 200µM GTP
for 15 min at 37°C in the presence of 250 mM NaCl or 250 mM KCl. Stimulation of GTPase
activity by the ribosome was tested by incubation of 100 nM of free 50S subunits with 100 nM
RbgA and 200 µM GTP for 15 min at 37°C in the presence of 250 mM NaCl or 250 mM KCl.
The activity was determined by standard malachite green assay as described in the materials and
methods. Data for three biological replicates with standard deviation of the mean presented.

47

Figure 2.1 Kinetic analysis of GTP hydrolysis rates by RbgA proteins.
GTP hydrolysis rates of RbgA proteins were determined by monitoring release of free phosphate
using malachite green/ammonium molybdate colorimetric assay as described in materials and
methods. Reactions were carried out under initial rate conditions (less than 10% of substrate
consumed). Values of kcat and Km were determined by fitting the Michaelis-Menten equation
using nonlinear regression algorithms provided by the GraphPad Prism software (GraphPad
Software, Inc.). Representative GTP hydrolysis curves of RbgA wild type ( ● ) and the P-loop
variant S134A ( ■ ) are shown. Error bars represent standard deviation of the mean of three
technical replicates.

48

Figure 2.2 Stimulation of GTPase activity of RbgA by ribosomal particles.
Stimulation of RbgA GTPase activity by ribosomes was assessed by determining the GTP
hydrolysis rates of RbgA in the presence of various purified ribosomal particles. 100 nM of
purified ribosome was incubated with 100 nM RbgA for 15 min at 37°C in the presence of
various concentrations of GTP and assayed as described in the Materials and Methods section.
The representative curves are of GTP hydrolysis rates determined based on the free phosphate
produced after the reactions had proceeded for 15 min at 37°C by reaction mixtures containing
RbgA only (●), RbgA and mature 50S (■), RbgA and free 50S (▲), or RbgA and 45S (▼). Error
bars represent standard deviation of the mean of three technical replicates.

49

Figure 2.3 Superimposition of MnmE and homology model of RbgA.
A. MnmE and RbgA homology model was superimposed using ligalign script in PyMOL which
align the two structures based on their ligand positions, which is GDP in this case. The K-loop of
MnmE (green) and RbgA (brown) occupies similar position (as shown in rectangle) around the
bound potassium in crystal structure. B. An enlarged view of catalytic pocket with GDP and
+
bound potassium is shown in which N130 from P loop of RbgA (brown) coordinates bound K
ion (indicated by purple ball) in similar fashion as N226 from MnmE (green) P loop (indicated
by arrows).

50

Figure 2.4 Interaction between RbgA and ribosome in the presence of different guanine
nucleotides.
Binding of RbgA to purified ribosomal subunits was tested by an in vitro binding assay in which
60 pmoles of RbgA was pre-incubated with 1.5 mM GDP, GTP, GMPPNP or pppGpp before the
addition of 10 pmoles of purified 45S or 50S ribosomal subunits. The mixtures were incubated
further for 15 min at 37°C then free RbgA was filtered off by centrifugation through a 100 kDa
cut-off Microcon columns. The columns were washed three times; first with buffer A and then
twice with buffer C (high salt buffer). RbgA-ribosome complexes were eluted and bound RbgA
was detected by immunoblotting using anti-RbgA antibody.

51

Figure 2.5 Proposed model for role of RbgA in 50S subunit maturation.
RbgA interacts with 45S in GTP bound form and introduces conformational changes that further
facilitate the binding of late ribosomal proteins like L16, L27 and L36 (depicted by different
ovals). Binding of these proteins promotes maturation of intermediate 45S subunit (white) to a
mature 50S subunit (gray). Along with the complete maturation of 50S subunit, GTP at RbgA is
hydrolyzed to GDP and this GDP bound RbgA and inorganic phosphate leave the mature 50S
subunit, which is now ready to take part in translation. It is also possible that RbgA completes
maturation of a late step that does not end in the final maturation of the 50S subunit.

52

ACKNOWLEDGEMENTS

We thank Marci Baranski and Loren Palmeri for technical assistance and Jade Wang (Baylor
College of Medicine) for providing pppGpp.

53

REFERENCES

54

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4. Culver, G.M. (2003) Assembly of the 30S ribosomal subunit. Biopolymers 68: 234-49.
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13. Campbell, T.L., Daigle, D.M., Brown, E.D. (2005) Characterization of the Bacillus subtilis
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15. Jensen, B.C., Wang, Q., Kifer, C.T., Parsons, M. (2003) The NOG1 GTP-binding protein is
required for biogenesis of the 60S ribosomal subunit. J Biol Chem 278: 32204-11.
16. Kallstrom, G., Hedges, J., Johnson, A. (2003) The Putative GTPases Nog1p and Lsg1p Are
Required for 60S Ribosomal Subunit Biogenesis and Are Localized to the Nucleus and
Cytoplasm, Respectively. Mol Cell Biol 23: 4344-55.
17. Saveanu, C., et al. (2001) Nog2p, a putative GTPase associated with pre-60S subunits and
required for late 60S maturation steps. Embo J 20: 6475-84.
18. Matsuo Y, O.T., Loh Pc, Morimoto T, Ogasawara N (2007) Isolation and characterization of
a dominant negative mutant of Bacillus subtilis GTP-binding protein, YlqF, essential for
biogenesis and maintenance of the 50 S ribosomal subunit. J Biol Chem 282: 25270-7.
19. Charollais, J., Pflieger, D., Vinh, J., Dreyfus, M., Iost, I. (2003) The DEAD-box RNA
helicase SrmB is involved in the assembly of 50S ribosomal subunits in Escherichia coli. Mol
Microbiol 48: 1253-65.
20. Lin, B., Thayer, D.A., Maddock, J.R. (2004) The Caulobacter crescentus CgtAC protein
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21. Lanzetta, P.A., Alvarez, L.J., Reinach, P.S., Candia, O.A. (1979) An improved assay for
nanomole amounts of inorganic phosphate. Anal Biochem 100: 95-7.
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comparative protein modeling by MODELLER. Proteins 23: 318-26.
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24. Schrödinger, L. (Version 1.2r3pre) The PyMOL 1.2r3pre ed. pp. Molecular Graphics System.
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26. Anand, B., Surana, P., Prakash, B. Deciphering the catalytic machinery in 30S ribosome
assembly GTPase YqeH. PLoS One 5: e9944.
27. Ash, M.R., Maher, M.J., Guss, J.M., Jormakka, M. The initiation of GTP hydrolysis by the
G-domain of FeoB: insights from a transition-state complex structure. PLoS One 6: e23355.
28. Scrima, A., Wittinghofer, A. (2006) Dimerisation-dependent GTPase reaction of MnmE:
how potassium acts as GTPase-activating element. EMBO J 25: 2940-51.
29. Scheffzek, K., et al. (1997) The Ras-RasGAP complex: structural basis for GTPase
activation and its loss in oncogenic Ras mutants. Science 277: 333-8.
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30. Neidhardt, F.C., Ingraham, J.L., Schaechter, M. (1990) Physiology of the Bacterial Cell: A
Molecular Approach: Sinauer Associates.
31. Lopez, J.M., Marks, C.L., Freese, E. (1979) The decrease of guanine nucleotides initiates
sporulation of Bacillus subtilis. Biochim Biophys Acta 587: 238-52.
32. Schmeing, T.M., et al. (2009) The crystal structure of the ribosome bound to EF-Tu and
aminoacyl-tRNA. Science 326: 688-94.
33. Delivron, M.A., Robinson, V.L. (2008) Salmonella enterica serovar Typhimurium BipA
exhibits two distinct ribosome binding modes. J Bacteriol 190: 5944-52.

57

CHAPTER 3

Mutational analysis of the ribosome assembly GTPase RbgA provides insight into ribosome
interaction and ribosome-stimulated GTPase activation

The contents of this chapter were published in Gulati M., Jain N., Anand B., Prakash B and
Britton R.A. (2013) Mutational analysis of the ribosome assembly GTPase RbgA provides insight
into ribosome interaction and ribosome-stimulated GTPase activation. Nucleic Acids Res.
41(5):3217-27. MG and NJ collaborated on Figures 3.7 and 3.8 and Table 3.2

58

Abstract
Ribosome biogenesis GTPase A protein (RbgA) is an essential GTPase required for the
biogenesis of the 50S subunit in Bacillus subtilis. Homologs of RbgA are widely distributed in
bacteria and eukaryotes and are implicated in ribosome assembly in the mitochondria,
chloroplast and cytoplasm. Previous work has shown that cells depleted of RbgA accumulate an
immature large subunit that is missing key ribosomal proteins. RbgA, unlike many members of
the Ras-superfamily of GTPases, lacks a defined catalytic residue for carrying out GTP
hydrolysis. To probe RbgA function in ribosome assembly we used a combined bioinformatics,
genetic and biochemical approach. We identified a RNA binding domain, ANTAR which are
known to bind structured RNA within the C-terminus of RbgA. Mutation of key residues in the
ANTAR domain altered RbgA association with the ribosome. We identified putative catalytic
residue His9 part of three amino acid motif PGH, which is similar to a domain within EF-Tu that
is directly involved in GTP hydrolysis upon interaction with the ribosome. Finally, our results
support a model in which the GTPase activity of RbgA directly participates in the maturation of
the large subunit rather than solely promoting dissociation of RbgA from the 50S subunit.

59

Introduction
Ribosomes are complex molecular machines that, in bacteria, contain three RNA molecules and
over 50 proteins [1-4]. The accurate and timely assembly of ribosomes is critical for organisms
to achieve the fast growth rates observed [4]. Though active ribosomes can be generated in vitro
without the need for additional assembly factors, non-physiological temperatures and salt
conditions are required [1,2,5]. In light of this observation Nomura and co-workers suggested
that ribosome assembly factors would be required in vivo at key steps that required these nonphysiological conditions in vitro. However, these assembly factors have remained elusive in
bacteria [4,6]. Over the past decade studies have shown that several GTPases likely play a
critical role in ribosome assembly (RA-GTPases) in both bacteria and eukaryotes [6-8]. However
the precise molecular function carried out by RA-GTPases during in vivo ribosome assembly
remains unknown.
RbgA (ribosome biogenesis GTPase A) is an essential, widely conserved GTPase that is required
for the assembly of the 50S subunit in Bacillus subtilis [9-12]. RbgA homologs are widely
distributed evolutionarily and eukaryotic homologs of RbgA such as Mtg1 (mitochondria), Lsg1,
Nug1, and Nog2 have been implicated in ribosome assembly [13-16]. RbgA proteins have
several unique features that distinguish them from conventional Ras like GTPases. First, RbgA
belongs to a family of circularly permuted GTPases (cpGTPases) in which the order of the
highly conserved G motifs (G1-G4) is altered such that the G4 motif precedes the G1 motif in
the sequence of the protein [17]. Although the reason for this circular permutation of the G
domain is unclear, in almost every case cpGTPases contain a second protein domain directly
downstream of the Switch II (G3) region. This would allow guanine nucleotide dependent
movement of this second protein domain, which is likely involved in binding to RNA or

60

interacting with ribonucleoprotein structures [17]. Secondly, RbgA and its homologs are also
classified as hydrophobic amino acid substituted GTPases (HAS-GTPases) [18]. HAS-GTPases
are involved in a wide variety of cellular functions such as ribosome biogenesis, tRNA
modification and cell cycle regulation [18]. In this family of GTPases the catalytic glutamine that
is usually present in Ras-like GTPases is replaced by a hydrophobic amino acid. Several
prokaryotic GTPases such as FeoB, MnmE, YphC YqeH, Obg and Era fall into this family [1925]. These GTPases utilize alternative mechanisms for GTP hydrolysis that have been deduced
with the help of crystal structures in two cases, MnmE and FeoB, but not for RbgA [26,27].
Cells depleted of RbgA do not form mature 50S subunits but rather accumulate a novel 45S
complex lacking three ribosomal proteins – L16, L27 and L36 [9,11]. In yeast, the RbgA
homolog Lsg1 is proposed to participate in the association of Rpl10p (L16 homolog) with the
60S subunit at a late stage during large subunit biogenesis [14]. Incorporation of L16 is also
predicted to occur at a late stage of 50S assembly and is accompanied by a large conformational
change in vitro [28], suggesting that RbgA proteins regulate an evolutionarily conserved step in
ribosome assembly. The GTPase activity of RbgA is stimulated ~60-fold by 50S subunits and
physical association with 50S subunits is maximal in the presence of a non-hydrolyzable analog
of GTP [12], indicating that GTP hydrolysis is responsible for dissociating RbgA from the
ribosome. Interaction with the 45S yields only a mild increase in GTPase activity and the
association of RbgA with 45S subunits is equal in the presence of GTP or a non-hydrolyzable
analog of GTP [12]. These and other observations have led to a model in which RbgA functions
by interacting with the large ribosomal subunit prior to the incorporation of L16 and either
directly recruits L16 to the ribosome complex or indirectly facilitates L16 binding by remodeling

61

the ribosome [12]. The molecular basis of RbgA mediated ribosome assembly, in particular the
role of specific amino acid residues, is poorly understood.
To elucidate how RbgA participates in ribosome assembly we utilized a site-directed
mutagenesis approach combined with in vivo and biochemical characterization of RbgA mutants.
We have discovered a RNA binding motif that mediates, in part, RbgA interaction with the 50S
subunit. We also identified a potential catalytic residue that mediates GTP hydrolysis. We
discuss our findings in relation to other ribosome associated GTPases and suggest that future
studies of RbgA in bacteria will also provide additional insight into mitochondrial and eukaryotic
ribosome assembly.

Materials and Methods
Growth conditions
All strains were grown at 37°C in LB (lysogeny broth) medium. Antibiotics were added at the
following concentrations when required: chloramphenicol (5 µg/ml), kanamycin (25 µg/ml),
spectinomycin (100 µg/ml) and ampicillin (100 µg/ml). In addition IPTG was added at a
concentration of 1 mM unless mentioned otherwise and xylose was added at a concentration of
2% when needed.

Construction of strains
All strains used in this study were derived from wild type Bacillus subtilis strain JH642 (RB247).
Strain RB301 was constructed as described [9]. Strain RB562 was constructed by switching the
antibiotic resistance cassette linked to Pspank and rbgA in RB301 from chloramphenicol to
spectinomycin. RB611 was constructed by transforming RB562 with plasmid pMAP65 [29] that
62

overexpresses lacI repressor to control the expression of wild type rbgA gene under the control
of Pspank promoter. Strain RB613 was constructed by cloning wild type rbgA into the pSWEET
plasmid [30] with a chloramphenicol resistance cassette and rbgA under the control of the PxylA
promoter. This construct was linearized and inserted at the amyE locus in RB611, creating strain
RB613. Chloramphenicol-resistant colonies were confirmed to have lost the ability to degrade
starch, indicating disruption of the amyE gene. The desired mutations were introduced into the
pSWEET plasmid using the QuikChange II XL kit (Stratagene) by following the manufacturers
instructions. The resultant plasmids with mutated rbgA gene were linearized and transformed in
RB611 strain. Resultant strains are dependent on IPTG for growth and are listed in Table 3.1.
Escherichia coli BL21(DE3) cells transformed with plasmid pET21b containing full-length rbgA
placed under the control of the T7 promoter were used to overexpress RbgA proteins [9]. Desired
mutations were constructed using the QuikChange II XL kit (Stratagene) by following the
manufacturers instructions.

Characterization of association between RbgA or mutants and 45S or 50S complexes
Wild type and mutant proteins were purified as described [12]. 50S subunits and 45S
intermediates were prepared by sucrose density gradient [12]. 50S and 45S complexes were
isolated from lysates of RB418 and RB301 cells respectively. The cells were grown were grown
to OD600 of 0.5 at 37°C in LB medium with or without IPTG. RB301 and RB418 cells were
grown without IPTG for several generations to deplete the cells of RbgA and IF-2, respectively,
until a doubling time of about 150 min was reached. Chloramphenicol (Sigma) was added to a
final concentration of 100 μg/ml 5 minutes prior to harvesting. 50S subunits and 45S complex

63

were isolated as described [12]. The association assay was performed with varying levels of
RbgA protein to 45S or 50S to determine the linear range of the assay. At each ratio the
nucleotide concentration was 400 µM, which is well above the Km of RbgA to ensure maximal
RbgA protein was bound to the nucleotide. All assays reported here were performed at 37°C
with 50 nM RbgA or mutant protein, 10 nM 45S or 50S and 400 µM nucleotide (GDP, GTP or
GMPPNP). The binding protocol was used as described [12]. Briefly, 60 pmol of RbgA was preincubated with 1.5 mM of different guanine nucleotides for 15 min followed by addition of 10
pmol purified subunits. The binding was allowed to proceed for 15 min at 37°C, then centrifuged
once through Microcon 100 filters (Millipore) with a cut-off of 100 kDa. The RbgA-subunit
complexes were washed once with buffer A, twice with buffer C before elution. Four controls
were performed for every assay - 45S/50S with no RbgA/mutant protein and RbgA/mutant
protein with each of the three nucleotides but no subunit. This was to ensure that no RbgA was
bound to the subunit while preparation of subunits and RbgA/mutant protein was not retained on
the filter in the absence of subunit respectively. The subunit bound RbgA was detected by
separating the complex on SDS-PAGE gels (12% Bis-Tris NuPAGE gel, Invitrogen) followed by
western blot analysis using polyclonal rabbit anti-RbgA and HRP-conjugated goat anti-rabbit
polyclonal antibodies (PerkinElmer Life Sciences). The bands were visualized using Western
Lightening chemiluminescent detection system (PerkinElmer Life Sciences). A series of
incremental exposures was obtained to determine that chemiluminescent signal was in the linear
range of detection. A single time point from the linear range was chosen and mutant and wild
type protein, present on the same blot membrane was quantified using FujiFilm Multi Gauge
v3.0 software. The binding level of RbgA/mutant protein was determined and the data is reported

64

as a percentage of wild-type RbgA association with either the 45S or the 50S subunit in the
presence of GMPPNP, which displays maximal binding.

Characterization of GTPase activity of RbgA/mutants
The assay was performed as described [12]. Briefly for measuring GTPase activity in the
presence of 50S subunits 100 nM RbgA/mutant protein was incubated with 100 nM 50S subunit
and 200 µM GTP at 37°C for 30 minutes and for measuring intrinsic GTPase activity 2 µM
RbgA/mutant protein was incubated with 200 µM GTP at 37°C for 30 minutes. We
predetermined that under these conditions the values were in the linear range of the assay. The
phosphate released was measured by malachite green colorimetric system (BioAssay systems).
The mutant proteins were assayed on the same 96 well plate (Corning) as the wild type protein.

Bioinformatics analysis and superimposition
Coordinates from the C-terminus of RbgA were used to search for similar structures using the
DALI server (http://ekhidna.biocenter.helsinki.fi/dali_server/) [31] and resulting structures were
superimposed using PyMol [32]. Superimposition of free EF-Tu (PDB: 1EFT) and ribosome
bound EF-Tu (PDB: 3FIC) were superimposed on RbgA (PDB:1PUJ) using ligalign script [33]
in PyMol. All the figures were made in Chimera [34].

Results
Multiple sequence alignment of RbgA homologs revealed 4 regions of high conservation
RbgA contains two structural domains, an N-terminal circularly permuted G-domain and a Cterminal domain that contains no sequence similarity to any known functional protein domain.

65

To identify key residues within conserved regions in RbgA we generated a multiple sequence
alignment of bacterial RbgA homologs using CLUSTAL-W (Figure 3.1A). In addition to the
universally conserved G-domain motifs G1-G4 we identified four regions of high conservation
designated CR1-CR4 (Figure 3.1A). Although these conserved regions are dispersed throughout
the protein sequence, they lie in close vicinity in the crystal structure of the protein (Figure
3.1C). The first two regions, CR1 and CR2 are contained in the N-terminal domain of RbgA and
precede the circularly permuted G-domain. Of these two CR1 was of particular interest as this
stretch of 15 amino acids found at the N-terminus of RbgA is largely conserved among all
bacterial RbgA homologs as well as eukaryotic Mtg1 proteins (Figure 3.1B). Most of CR1 is
unstructured in all available RbgA crystal structures deposited to date, indicating its dynamic
nature and thus its role in RbgA function cannot be assessed by its structure. CR3 is a highly
conserved loop that links the N-terminal domain to the C-terminal domain and contains the
Switch II motif. Thus it is likely to participate in communication between the N and C terminal
domains when bound to either GTP or GDP. The C-terminal domain following the CR3 showed
low sequence similarity and only one moderately conserved region (CR4) was identified in this
domain.

The C-terminal domain of RbgA contains an ANTAR RNA binding domain
Because the C-terminal domain of RbgA contained low sequence similarity to proteins of known
function in the NCBI database (other than RbgA homologs) we asked if the C-terminal domain is
structurally similar to any previously characterized domains. To identify proteins that contain a
structurally similar domain, we used the structure of only the C-terminal domain (177-269
residues) of RbgA as a query to search the DALI database [31]. The first 4 hits (Table 3.2) were

66

from crystal structures of either RbgA or its homologs as expected (DALI Z-score= 7.3-14.8,
rmsd=0-2.8 Å). The DALI score provides a measure of structural similarity between a given pair
of protein domains and structures that have significant similarities have a Z-score greater than 2.
The first non-RbgA hit was the C-terminal domain of a NasR, a protein that mediates
transcription anti-termination (Z =4.1, rmsd=3.6 Å) [35]. The next few hits were with incomplete
domains of exodeoxyribonuclease and Cdc4, followed by other potential transcription antiterminators including AmiR [36]. These results showed that the C-terminal domain of RbgA had
significant structural similarity to the C-terminal domains of two proteins that are involved with
promoting transcription antitermination, AmiR and NasR (Table 3.2). This C-terminal domain
present in both AmiR and NasR interacts with structured RNA and encompasses a RNA binding
domain denoted the ANTAR (AmiR-NasR Transcription Anti-termination Regulators) domain
[37]. The ANTAR domain, characterized by a three-helix structure, is structurally similar to the
C-terminal domain of RbgA (Figure 3.2) [37]. Superimposition of the RbgA C-terminal structure
on the C-terminal regions of NasR and AmiR shows a topologically similar three-helix
arrangement, typical of ANTAR domains (rmsd=3.6 and 3.4 Å respectively) (Figure 3.2). It is
noteworthy that the structural similarity notwithstanding, the sequence similarity of these
proteins with the RbgA C-terminal domain is remarkably low (13% for NasR and15% for
AmiR).

Site-directed mutagenesis and phenotypic analysis of RbgA mutants
Since rbgA is an essential gene, null mutations will not survive and cannot be studied. Therefore,
we developed a strain that allowed mutations that negatively affect RbgA function to be
generated and characterized. A conditional rbgA mutant strain was created by placing a wild-

67

type copy of the rbgA at its native locus under the control of the Pspank promoter, yielding a
strain that is dependent on IPTG for growth. Mutant versions of a second copy of the rbgA gene
were placed under the control of the Pxyl promoter and introduced at the amyE locus. The
expression of the mutant rbgA gene is therefore dependent on the presence of xylose in the
growth media. In order to study mutations in rbgA we constructed strains in the presence of
IPTG and then interrogated their phenotype by growing cells in the presence of xylose and
absence of IPTG. As a control we created a strain (RB613) that contains a wild-type copy of the
rbgA gene under the control of the Pxyl promoter at the amyE locus. As expected, RB613 grows
equally well in the presence of 1 mM IPTG or 2% xylose (data not shown).
Using this system we screened 44 mutations in rbgA to assess their growth phenotypes by
streaking each mutant rbgA strain on plates containing either 1 mM IPTG or 2% xylose. A
representative result is shown in Figure 3.3. Previous work on GTPases has shown that mutating
the S/T residue in the P-loop (motif G1 -GXXXXGKS/T) abolishes GTP hydrolysis [38]. We
constructed the corresponding RbgA mutant strain (S134A) and found that this mutation was
lethal and no colonies were observed on plates containing 2% xylose, in contrast to the control
strain RB613 (Figure 3.3C). Both strains displayed wild-type growth when streaked on plates
containing 1 mM IPTG (Figure 3.3B) while no growth was observed in the absence of either
inducer (Figure 3.3D). Using this system we identified 17 mutations that negatively affected cell
growth (Figure 3.4). We further quantified the effect of each mutation by measuring growth rates
in liquid media. Mutations that reduced the growth rate 3 fold or less compared with wild type
growth were classified as causing a mild growth effect while mutations that reduced cell growth
more than 3 fold were classified as severely affecting cell growth. We found that 12 mutations

68

cause a severe growth defect while 5 mutations have a milder effect on cell growth (Table 3.3
and Figure 3.5). To verify that defective RbgA proteins that did not support growth were able to
accumulate inside the cell, we confirmed that the expression of all mutant RbgA proteins was
similar to wild-type expression levels with the exception of one case (D228A/E229A) (data not
shown). Mutations that alter the CR1, CR3 and ANTAR residues and that caused a severe
growth defect were characterized further and are discussed below.
In constructing our strains we did not find any indication of a strong dominant negative effect of
any mutation in RbgA. Previously it was reported that a deletion of the first 10 amino acids of
RbgA (referred to as ∆N10) resulted in a dominant negative phenotype [39]. We tested all of our
mutants and the∆N10 RbgA mutant for dominant negative effects using 1 mM IPTG and 2%
xylose. Under these conditions none of the mutations tested displayed any phenotype, including
∆N10, as all cells grew at a wild-type growth rate. Reduction of IPTG levels to 100 µM, which
reduces expression of the wild-type copy of rbgA, still allows cells to proliferate at wild-type
growth levels.

Growing cells with 100µM IPTG and 2% xylose uncovered the dominant

negative phenotype of the∆N10 mutant. Under these conditions we identified three additional
mutations in our collection that also had dominant negative effects, F6A and H9A in CR1 and
Y225A in the ANTAR domain (Table 3.4). We note that when cells are grown under 100 µM
IPTG and 2% xylose the protein expression level of mutant copy of RbgA is at least 10-fold
higher than the wild-type copy, indicating that these RbgA mutations have a weak dominant
negative phenotype.

69

Biochemical characterization of RbgA mutants
In order to fully characterize the effects of mutations that were found to be severely deleterious
to growth we A) monitored the ability of RbgA mutants to associate with both 50S subunits and
45S complexes and B) assessed their ability to catalyze GTP hydrolysis in the presence and
absence of 50S subunits. The results of these two experiments are discussed in the following two
sections. Seven mutants were selected for these studies based on their location in the protein and
their phenotype of severely reduced growth rates relative to wild-type RbgA. Three are from
CR1 (F6A, H9A, K12E/R14E), one from CR3 (F180A), and three (A206D, Y225A, I241D)
from the ANTAR domain. These RbgA mutants were purified as C-terminal His-tag fusions;
previous work has shown that wild-type RbgA with a C-terminal His-tag is fully functional [9].

Mutations in the ANTAR domain alter RbgA:ribosome association
We have previously shown that RbgA stably binds to the 45S complex and 50S subunit in a
nucleotide-dependent manner [12]. RbgA binds to the 50S subunit in the presence of a nonhydrolyzable GTP analog, GMPPNP, and binding in the presence of GTP is reduced, likely due
to GTP hydrolysis upon interaction with the 50S subunit [12]. In contrast, both GTP and GMPPNP enhance binding of RbgA to the 45S complex equally [12]. We hypothesized that the
ANTAR domain of RbgA is essential for association with the ribosome and targeted five key
structural residues in the ANTAR domain for mutation as described below.
The ANTAR domain comprises three alpha-helices and is stabilized by coiled-coil interactions
(Figure 3.2).

ANTAR domains display low sequence conservation amongst homologs and

contain only five strictly conserved residues which have their side-chains exposed into the cavity
formed between the three helices and hence stabilize the structural fold [37]. RbgA contains four

70

of these conserved residues at the corresponding position in the domain: A206, A235, F238 and
I241. Our results show that substitutions in two of these residues (A206D and I241D) caused a
severe growth defect, while substitutions in A235 or F238 had no effect on growth (Figure 3.4
and Table 3.3). We also generated a fifth mutant, Y225A, which likely disrupts non-covalent
interactions between the helices and may alter the structure of the domain. Y225A caused a
severe growth defect and displayed a weak dominant negative phenotype (Figure 3.4 and Table
3.4). We proceeded to test association of mutated RbgA proteins with the 45S intermediate and
the 50S subunit under different nucleotide conditions using a quantitative in vitro binding assay.
Purified RbgA proteins were incubated with either purified 50S subunits or 45S intermediates for
15 minutes at 37°C. These complexes were then centrifuged through a 100 kDa Millipore filter
to remove unbound RbgA from the sample. The remaining RbgA that is associated with the
ribosomal subunit was then analyzed and quantified by Western blot analysis. A representative
example of the results is depicted in Figure 3.6. Wild-type RbgA binds most efficiently to the
50S subunit in the presence of GMPPNP, with a ~5-fold reduction in association in the presence
of GTP and ~30-fold reduction in the presence of GDP. A206D, a strictly conserved residue in
the ANTAR domain renders RbgA unable to associate with the 50S subunit (Figure 3.6) or 45S
intermediate (Table 3.5) under any nucleotide condition. Since A206D retains some GTPase
activity (see below), we do not expect that the lack of binding observed is solely due to complete
loss of the tertiary structure of the entire protein.
The binding of each RbgA mutant protein was determined in a similar manner and the results are
reported as a percentage of wild-type RbgA association with either the 45S or the 50S subunit in
the presence of GMPPNP, which displays maximal binding. Notably, of the eight mutant
proteins tested, the most striking results were seen with substitutions in the ANTAR domain that

71

changed the binding of RbgA to the 50S or 45S subunits. Mutations in CR1 were only altered in
GTP bound form, a result of the altered GTPase activity of these mutants which will be
discussed in the next section and mutation in CR3 displayed near wild-type levels of binding and
retained the same nucleotide dependence as wild-type RbgA. In contrast, all three mutants
targeting the ANTAR domain displayed altered binding and surprisingly had different
phenotypes. A206D completely lost all ability to stably associate with the ribosomal subunits,
while I241D displayed reduced association (~10 fold) with the 50S or 45S subunits in the
presence of GTP. The most surprising result was observed with the Y225A mutant RbgA
protein. This RbgA derivative was greatly reduced in its association with the subunits in the
presence of GMPPNP, displaying binding levels comparable to those observed with GDP
(Figure 3.6). However, Y225A associated with both the 50S and 45S subunits in the presence of
GTP at higher levels than observed with the wild-type RbgA protein, indicating that this
mutation may stabilize interaction in state of partial GTP hydrolysis (Figure 3.6). Thus all three
ANTAR domain mutants display altered association with both the 45S and 50S subunits,
highlighting the importance of this domain in association with the ribosomal subunit.

Mutations in CR1 severely reduce GTPase activity of RbgA
RbgA has a low intrinsic GTPase activity which is stimulated ~60 fold in the presence of mature
50S subunits [12]. We therefore assessed the ability of the 50S subunit to stimulate the GTPase
activity of RbgA mutants. As a negative control we utilized a mutant protein containing the
S134A substitution in the P-loop, which shows no detectable intrinsic GTPase activity and is
inert to simulation in the presence of the 50S subunit. Our results show that mutations in CR1
and the ANTAR domain impact the GTPase activity of RbgA, while the mutation in CR3 had no

72

effect (Table 3.6). However, mutations in the ANTAR domain also disrupted interaction of
RbgA with both the 45S as well as the 50S subunit and therefore the attenuated GTPase activity
seen in A206D and I241D is likely due, in part, to an indirect effect of poor association with the
50S subunit (Table 3.5, and discussion above).
Mutations in CR1 had a strong impact on the GTPase activity of RbgA. Mutation F6A resulted
in a ~11 fold reduction in intrinsic GTPase activity and was stimulated ~15 fold in the presence
of the mature 50S subunit, demonstrating that this mutant is reduced but still capable of GTP
hydrolysis. Mutation K12E/R14E had a moderate reduction in intrinsic GTPase activity but was
stimulated only 2-fold in the presence of the 50S subunit. These results suggest that the flexible,
highly conserved N-terminal region of RbgA encompassing CR1 is required for GTPase
activation in the presence of the ribosomal subunit.
A recent crystal structure of EF-Tu in complex with the ribosome demonstrated that interaction
between the ribosome and His84 of EF-Tu was required for efficient hydrolysis of GTP [40].
Voorhees et al. noted that His84 is preceded by a proline and glycine (denoted the PGH motif),
which were proposed to be critical for the correct positioning of His84 upon interaction with
nucleotide A2662 of the ribosome during activation. We observed that the CR1 region of RbgA
also contains a PGH motif (amino acids 7-9) that is highly conserved in bacterial and eukaryotic
RbgA homologs (Figures 3.1A and 3.1B). An alignment of crystal structures of RbgA and free
EF-Tu indicate that His9 from PGH motif of RbgA is in a similar position as the catalytic His84
of EF-Tu (Figure 3.7 and Figure 3.8). To test the role of this histidine residue (His9) in the
GTPase activity of RbgA, we generated a H9A mutant and characterized its GTPase activity in
the absence and presence of the 50S subunit. Our results show that mutation H9A reduced
intrinsic GTPase activity of RbgA by ~6 fold relative to the wild-type protein. Most significantly

73

this mutant displayed no detectable GTPase activity in the presence of the 50S subunit (Table
3.6). Together this result, in conjunction with the phenotype of F6A and K12E/R14E, shows that
mutations in CR1 reduce GTPase activity of RbgA and that His9 is required for GTPase activity
in the presence of the 50S subunit. The absence of GTPase activity in the mutant is unlikely to be
due to reduced association with the 50S subunit as this mutant in particular and other CR1
mutants associated with the 50S subunit at similar levels compared to wild type RbgA (Table
3.5).

Linker region is required for RbgA function
All cpGTPases have a C-terminal domain linked to the G3 motif (Switch II) [17]. In RbgA a
highly conserved loop (CR3) links the C-terminal ANTAR domain to the G3 motif (Switch II).
We hypothesized that the CR3 could be crucial for communicating conformational changes
during GTP hydrolysis between the G3 motif and the ANTAR domain. We identified one
mutation in CR3, F180A that was lethal. We tested this mutation utilizing the in vitro binding
assay described previously to determine if this residue was required for association of RbgA to
the ribosomal subunits and found that this mutation did not alter association of RbgA to the 45S
complex or the 50S subunit (Table 3.5). Next, we characterized the GTPase activity of this
mutant and found that mutation F180A has a mild effect on GTPase activity of RbgA with and
without the 50S subunit (Table 3.6). Thus, even though this mutation is defective neither in
ribosome association nor in ribosome stimulated GTPase activity it is unable to support growth
and yields ribosome assembly defects in vivo.

74

Strains expressing mutated RbgA proteins do not accumulate a novel intermediate
A previously proposed model suggested that GTP bound RbgA binds to the 45S intermediate and
either directly or indirectly recruits the ribosomal protein L16 to the intermediate [12]. Stable
integration of L16 and formation of a mature 50S subunit likely triggers GTP hydrolysis and
dissociation of RbgA from the mature 50S subunit. We hypothesized that strains that show a
growth defect phenotype due to the expression of a compromised RbgA protein may accumulate
a trapped intermediate that differs from the 45S intermediate observed upon depletion of the wild
type protein. We analyzed ribosome profiles of strains expressing RbgA mutants that negatively
affected cell growth and isolated the 45S intermediate from each mutant (a representative set is
shown in Figure 3.9). Our results show that strains expressing mutated RbgA proteins do not trap
a new intermediate. These strains also accumulated the 45S intermediate similar to RbgA
depleted cells and this intermediate also lacked the ribosomal protein L16 (Figure 3.10). Thus,
no novel ribosomal intermediate could be detected for the RbgA mutants that were analyzed.

Discussion
Ribosome assembly GTPases (RA-GTPases) are critical for the efficient and accurate biogenesis
of cellular, mitochondrial, and chloroplast ribosomes; however their roles in the assembly
process are poorly understood [4,6,8]. RbgA, and its closest eukaryotic homolog Mtg1, are
involved in the assembly of the large ribosomal subunit in bacteria and mitochondria,
respectively [9,11,13]. RA-GTPases use a variety of RNA binding domains to interact with RNA
including OB-folds (Obg) and KH-domains (Era) [24,41]. The crystal structure of RbgA showed
that the C-terminal domain (201-269) is connected to the N-terminal G-domain (1-176) through
a linker helix (177-200) (Figure 3.2). We have identified a novel RNA binding domain within

75

the C-terminus of RbgA, the ANTAR domain, which was discovered in proteins that bind
structured mRNA leader regions and promote transcription antitermination [37]. ANTAR
domain containing proteins such as NasR and EutV promote antitermination by interacting with
mRNA and promoting the formation of stem-loop structures that preclude the formation of
intrinsic transcription terminators [35,42,43]. It is attractive to speculate that RbgA may
influence ribosome assembly by interacting with rRNA via its ANTAR domain and stabilizing
the formation of a critical stem-loop in the 23S rRNA that does not form spontaneously and
prevents the formation of other nonproductive loops that may hamper further assembly. It is
possible that in in vitro experiments these nonproductive loops are circumvented by high
temperature or high salt conditions thereby facilitating proper assembly. Efforts to identify the
RbgA binding site on the ribosome are currently underway. Mutations in the ANTAR domain
reduced association of RbgA to the 45S or 50S to varying degrees. Residue A206 is likely
important for the structural stability of the ANTAR domain. This residue is one of four highly
conserved alanine residues seen in most ANTAR structures [37]. In addition mutants such as
Y225A could prove to be good candidates for structural and CryoEM studies as they show a
unique binding profile compared to wild-type RbgA and could shed light on the interaction of
RbgA with the 50S subunit.
Most ANTAR domain proteins are two domain proteins in which the ANTAR domain is
precluded from interacting with RNA until a signal or ligand is sensed by the receiver domain,
which opens up ANTAR for RNA binding [42,43]. One example of this arrangement is seen in
the recently published NasR structure where the signal receiver domain (NIT) and the ANTAR
domain are joined by a linker [35]. Thus, a similar arrangement in RbgA suggests that the
guanine nucleotide dependent conformational change of the N-terminal G-domain could be

76

communicated to the ANTAR domain through the conserved linker region. In principle, the
mechanistic reverse wherein binding of the ANTAR domain to rRNA could be coupled to a
conformational change in the N-terminal G-domain that facilitates GTP hydrolysis could also
occur. In both scenarios communication between the N- and C- terminal domains is important
for RbgA function and would likely rely on the linker CR3. Our results indicate that mutation in
this region is deleterious to growth though neither the association with subunit nor the GTPase
activity of this mutant protein is affected suggesting that while the function of either domain is
not lacking when tested independently the mutant protein cannot function in vivo.
The GTPase activity of RbgA is essential to its function and mutations in conserved G-domains
that abolish GTP hydrolysis are lethal.. However due to the absence of a crystal structure of
RbgA bound to 50S the exact mechanism of GTP hydrolysis remains elusive. Recent structural
evidence demonstrating how EF-Tu carries out GTP hydrolysis upon interaction with the
ribosome during accommodation has shown that positioning of His84 via interaction with 23S
rRNA nucleotide A2662 is critical for GTP hydrolysis [40]. His84 is part of a PGH motif that is
conserved in nearly all EF-Tu homologs identified to date and a similar PGH motif is found in
bacterial and eukaryotic homologs of RbgA. A key difference between the conserved PGH
motifs in EF-Tu and RbgA is the fact that whereas His84 is found in the Switch II (G3) region
(His84 substitutes for the catalytic Gln normally found in Ras-superfamily GTPases), His9 in
RbgA is found in the highly flexible CR1 region and is not ordered in the available crystal
structures (1PUJ) [44]. In RbgA the Switch II region is instead linked to the C-terminal ANTAR
domain. Mutation of His9 of RbgA resulted in a severely attenuated intrinsic rate of GTP
hydrolysis and no stimulation of GTPase activity in the presence of the 50S subunit could be
detected. Importantly this mutation had no significant effect on association with the 50S subunit.

77

Mutations in other parts of CR1 also greatly impacted GTP hydrolysis, however all other
mutations showed at least some stimulation in the presence of the 50S subunit. Our data is
consistent with a model in which the highly flexible CR1 region, upon interaction with the
ribosome, adopts a conformation that suitably positions His9 for GTP hydrolysis.
This assertion is also supported by analysis of the EF-Tu crystal structure [40]. Vorhees et al.
suggested that the EF-Tu GTP hydrolysis mechanism could be a general mechanism used by
translation GTPases to carry out GTP hydrolysis. To this end we superimposed the G-domain of
RbgA and EF-Tu (Figure 3.7 and Figure 3.8) by utilizing the GDPNP bound to each of these
proteins in the crystal structure as a point of reference. In the superimposed structure, His9 from
RbgA is in close proximity to His84 in the structure of free EF-Tu consistent with our assertion
above (Figure 3.7 and Figure 3.8). It should be noted that the position of catalytic His84 in EFTu is different in the free EF-Tu structure compared to the ribosome bound structure (Figure 3.7
and Figure 3.8). Since only the structure of free RbgA is available it is difficult to predict if His9
would move to the exact position as EF-Tu His84 upon interaction with the ribosome and if it
would interact with the same 23S rRNA nucleotide A2662. It is also noteworthy that due to
relocation of the putative catalytic residue His9 in RbgA from Switch II to the N-terminal helix,
a direct interaction between histidine and the activated water positioned to attack the γphosphate, requires a small translocation of Switch II and potentially the ANTAR domain as
well.
Based on previously published mechanisms of GTP hydrolysis this can be accomplished by one
of the following ways. First, binding of the ANTAR domain to the 50S subunit could result in a
conformational change that is transmitted through the linker CR3 to Switch II. This would likely
be coupled with the rearrangement of the N-terminal helix with concomitant positioning of His9

78

that would facilitate catalysis. The presence of a dynamic and unstructured N-terminal CR1 as
evidenced by relatively high B-factors of this region in the RbgA crystal structure, as well as a
flexible linker domain connecting Switch II to the ANTAR domain, further strengthen this
possibility. A nucleotide dependent large domain rearrangement has also been shown to occur in
other ribosomal GTPases such as YphC [21]. A second possibility is that the histidine residue
might take part in catalysis through additional intermediate water molecule(s), as has previously
been shown for other HAS-GTPases (MnmE and FeoB) [26,27]. In these GTPases the catalytic
residue interacts with a secondary (in the case of MnmE or tertiary in the case of FeoB) water
molecule that in turn activates the catalytic water molecule and results in GTP hydrolysis
[26,27]. Further, it is also possible that the mechanism in RbgA might be a combination of both
these mechanisms. Additional structural and biochemical studies are required to test either of
these mechanisms and to further ascertain if the nucleotide equivalent to A2662 in B. subtilis is
required for hydrolysis. This will require the development of tools to isolate mutant ribosomes
from this organism similar to the tools that have been developed for E. coli [45,46]. The GTPase
activity of RbgA was unaffected by E. coli 50S ribosomes, which was not unexpected given that
E. coli lacks a RbgA homolog and thus we could not test the interaction of RbgA with A2662
from the E.coli 50S subunit.
Although we have uncovered important insights into the biochemistry of RbgA and how RbgA
interacts with the ribosome, several important questions regarding RbgA (and RA-GTPases in
general) remain. First, does the GTPase activity of RbgA play a direct role in the assembly
process? If the GTPase activity was solely involved in displacing RbgA from the ribosome once
assembly had been completed then we expected to identify RbgA mutations that completed
assembly but not dissociate from the ribosome. This was not the case as all mutations that were

79

deleterious to growth yielded a 45S complex that lacked L16, which supports a direct role for
GTP hydrolysis in the assembly process. Second, what part of the 23S rRNA does RbgA interact
with during the assembly process? At this point there is limited chemical footprinting data that
shows RbgA interacting with the central protuberance [11]. We are currently mapping the
structure of the 23S rRNA in the 50S subunit and 45S complex to identify key sites that are
altered in the 45S complex. In addition, since RbgA homologs are not found in E. coli, we are
also targeting unique stem-loops within the B. subtilis 23S rRNA as possible sites where RbgA
may act. Third, does RbgA serve as a checkpoint to monitor large subunit assembly and correct
incorporation of L16?

Recent work in yeast has uncovered a novel type of translational

checkpoint utilized by ribosome assembly factors [47]. Finally, why do most organisms, ranging
from bacteria to humans, require RbgA to form ribosomes while many proteobacteria have lost
this requirement?

Future work will uncover the precise role of RbgA in the assembly of

ribosomes in all three kingdoms of life.

80

Strain
B. subtilis
RB301
RB419
RB611
RB613
RB667
RB720
RB653
RB633
RB750
RB752
RB754
RB744
RB655
RB654
RB635
RB708
RB656
RB711
RB657
RB622
RB756

Relevant genotype

Source

JH 642 Pspank-rbgA cat pMAP65
JH 642 Pspank-infB cat pMAP65
JH 642 Pspank-rbgA Spcr pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA cat pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-Q4L cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-F6A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-H9A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-K12E/R14E
cat pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-R14E cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-R15E cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-R34E cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-S38A/S39A
cat pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-K59A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-N58A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-D61A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-K107E cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-K107A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-K114E cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-K114E cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-R122A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-R122E cat
pMAP65

[9]
[9]
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Table 3.1 List of B. subtilis strains used in this study.
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Table 3.1 (cont’d)
RB1155
RB758
RB631
RB623
RB718
RB742
RB704
RB722
RB621
RB659
RB1153
RB658
RB851
RB863
RB617
RB865
RB921
RB853
RB819
RB821

JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-S134A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-T135A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-P129R cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-P129A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-T155A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-T155A/T155A
cat pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-F180A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-Q158A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-E181A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-W177F cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-W177A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-K196A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-A206D cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-Y225A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgAD228A/E229A cat pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-I241D cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-C247A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-A206L cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgAK244A/R245A cat pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgAK244A/R245A/R265A cat pMAP65

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Table 3.1 (cont’d)
RB823
RB825
RB619
RB855
RB857
RB859
RB861

JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgAK244A/R245A/K271A cat pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgAR224A/K244A/R245A/R265A cat pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgAE232A/D233A cat pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-A235D cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-A235L cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-F238A cat
pMAP65
JH 642 Pspank-rbgA Spcr amyE::Pxyl-rbgA-Y256A cat
pMAP65

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S. No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

Chain
1puj-A
3cno-A
3cnn-A
3cnl-A
4akk-A
2qxf-A
3hp9-A
1fxx-A
3c94-A
1nex-B
3c95-A
1sd5-A
2jvw-A
1s8n-A
1qo0-D
1ucp-A
4akk-B
1qo0-E

Z rmsd lalign
14.8
0
105
7.7
2.4
92
7.5
2.5
93
7.3
2.8
93
4.1
3.6
53
3.7
2.7
70
3.7
2.7
69
3.5
2.6
69
3.5
3
72

nres
261
227
227
233
368
433
452
459
458

%id
100
22
22
22
11
13
13
13
13

3.5

4

72

444

8

3.4
3.4
3.3
3.3
3.3

2.8
5.7
5
5.6
3.4

69
58
67
58
46

446
188
82
190
189

13
12
13
12
15

3.2

3.2

65

91

14

3.2
3.2

3.9
3.8

52
50

367
194

12
14

Description
conserved hypothetical protein Ylqf
putative uncharacterized protein
putative uncharacterized protein
putative uncharacterized protein
nitrate regulatory protein
exodeoxyribonuclease I
exodeoxyribonuclease I
exonuclease I
exodeoxyribonuclease I
centromere dna-binding protein
complex cbf3
exodeoxyribonuclease I
putative antiterminator
uncharacterized protein
putative antiterminator
AmiC
apoptosis-associated speck-like
protein
nitrate regulatory protein
AmiC

Table 3.2 Results from a search of structures similar to RbgA C-terminal domain in DALI
server.

84

Mutation
RB613
Q4L
F6A
H9A
K12E+R14E
K59A
K107E
K114E
P129R
S134A
T135A
T155A
F180A
A206D
Y224A
I241D
C247A

Domain

Doubling time in 2% xylose (minutes)
26 ± 1
66 ± 2
118 ± 4
138 ± 3
99 ± 2
105 ± 3
46 ± 2
44 ± 1
108 ± 3
148 ± 1
68 ± 3
104 ± 3
169 ± 3
109 ± 4
174 ± 4
114 ± 3
70 ± 3

CR1
CR1
CR1
CR1
G-domain

G-domain
G-domain
G-domain
CR3
ANTAR
ANTAR
ANTAR
ANTAR

Table 3.3 Growth rate of RbgA mutants.
Strains were grown in LB + 2% Xylose at 37°C. Strain RB613 is a control strain with wild type
rbgA gene under the control of Pxyl promoter. In the other strains mutated rbgA gene is under
control of Pxyl promoter. The data represents three independent growth experiments. Mutations
that reduced growth by 3-fold compared with wild type were classified as mild – these include
Q4L, K107E, K114E, T135A and C247A. The mutations that reduced the growth rate by more
than 3-fold were classified as severe – these include F6A, H9A, K12E/R14E, K59A, P129R,
S134A, T155A, W177A,F180A, A206D, Y225A and I241D.

85

Mutation

Domain

Wild type
F6A
H9A
Y225A

CR1
CR1
ANTAR

Doubling time in 100 µM
IPTG (minutes)
29 ± 2
31 ± 1
30 ± 2
30 ± 1

Doubling time in 100µM IPTG
+ 2% xylose (minutes)
26 ± 1
59 ± 4
62 ± 1
50 ± 2

Table 3.4 Dominant negative phenotype observed under specific growth conditions.
Strains were grown in LB + 1mM IPTG (full induction of wild type RbgA protein) and LB + 100
µM IPTG + 2% xylose to assess the dominant negative phenotype. The reduced 100 µM IPTG
reduces the wild type RbgA protein level and 2% xylose induces the expression of mutated
RbgA protein.

86

Domain/
Region

ANTAR

CR1
CR3

Mutation
RbgA wt
A206D
Y225A
I241D
F6A
H9A
K12E+R14E
F180A

50S
GDP*

GTP

5%
<0.6%
4%
1%
1%
1%
4%
6%

22%
<0.6%
380%
2%
68%
65%
64%
16%

GMPPNP
100%
<0.6%
5%
58%
71%
69%
67%
79%

45S
GDP

GTP

4%
<0.6%
7%
2%
2%
3%
2%
5%

84%
<0.6%
180%
5%
70%
69%
71%
86%

GMPPNP
100%
<0.6%
7%
68%
73%
72%
75%
91%

Table 3.5 Results for in vitro binding of RbgA mutants to the 45S and 50S subunits under
different nucleotide conditions.
The values represent the average of three independent experiments ± S.D.

87

Domain/
Region

Mutation

RbgA wt
F6A
CR1
H9A
K12E+R14E
G-domains
S134A
A206D
ANTAR
Y225A
I241D
CR3
F180A

Intrinsic
GTPase activity

Stimulation of GTPase
activity with 50S

V in pmol phosphate/ pmol RbgA/min
0.207 ± 0.008
12.57 ± 0.18
0.018 ± 0.002
0.283 ± 0.05
0.031 ± 0.006
<0.002
0.121 ± 0.004
0.226 ± 0.07
<0.002
<0.002
0.040 ± 0.001
0.978 ± 0.06
0.139 ± 0.005
3.97 ± 0.30
0.104 ± 0.007
0.597 ± 0.11
0.097 ± 0.012
8.08 ± 0.19

Fold
stimulation in
the presence
of 50S
61 ± 3
15± 4
2 ± 0.5
24 ± 2
29 ± 3
6 ± 0.5
84 ± 11

Table 3.6 Measurement of GTPase activity of RbgA mutants in the presence and absence of the
50S subunit.

88

A.

Figure 3.1 Sequence alignment of RbgA with its homologs.
A. Multiple sequence alignment of selected bacterial RbgA homologs. Highly conserved regions
are underlined. G-domain motifs are indicated as G4 (NKXD), G1 (GXXXXGKS/T), G2 (T),
and G3 (DXXG) in addition to four conserved regions (CR1-CR4).
89

Figure 3.1 (cont’d)
B.

B. Multiple sequence alignment of RbgA with eukaryotic homologs Mtg1 from humans and
mouse highlighting conserved region CR1. G-domain motifs are indicated G1-G4. Alignments
were constructed with ClustalW with default parameters and species indicated to the left. ‘*’
indicates positions which have a single, fully conserved residue, ‘:’ indicates conservation
between groups of strongly similar physicochemical properties, ‘.’ indicates conservation
between groups of weakly similar physicochemical properties.

90

Figure 3.1 (cont’d)
C.

C. Structure of RbgA depicting CR1, CR2, CR3 and CR4. Crystal structure of RbgA (PDB:
1PUJ) depicting the three conserved regions. First 9 amino acid residues are not structured in the
crystal. CR1 (9-17) is shown in red, CR2 (30-42) is shown in yellow, CR3 (175-182) is shown in
green and CR4 (188-197) shown in blue. All four conserved regions are dispersed across the
protein sequence and lie in close vicinity in the structure of the protein. For interpretation of the
references to color in this and all other figures, the reader is referred to the electronic version of
this thesis.

91

Figure 3.2 C-terminal domain of RbgA contains RNA binding domain ANTAR.
A. The crystal structure of RbgA (PDB: 1PUJ). The N-terminal G- domain is depicted in blue (9176), the C-terminal domain is depicted in red (201-282) and a linker helix connecting the two
domains is depicted in yellow (177-200). B. The superposition of the ANTAR domains of RbgA
(red), AmiR (green) and NasR (Cyan).

92

Figure 3.3 Testing the growth phenotype of RbgA mutants.
A. All strains were constructed with the wild-type rbgA gene under the control of an IPTG
inducible promoter and a mutated rbgA gene (S134A is shown as an example here) under the
control of a xylose inducible promoter. In contrast, the control strain had the wild-type rbgA
gene under the control of both promoters B. The two strains were streaked on IPTG containing
plates. Wild-type growth is seen in both strains due to the expression of rbgA. C. The two strains
were streaked on plates containing xylose. No colonies were observed for S134A and hence the
mutation was characterized as lethal. The RB613 control strain showed wild-type growth as rbgA
is expressed from xylose inducible promoter D. The two strains were streaked on plates without
IPTG or xylose. No growth is seen due to the absence of protein induction in either case.

93

Figure 3.4 Consolidated results of mutational analysis of RbgA.
Domains are indicated in grey. CR1-3; conserved region, G1-G4; conserved GTP binding
domains found in GTPase superfamily, ANTAR; AmiR-NasR transcription anti-termination
domain. The mutations shown in red are sick or do not support growth and the mutations shown
in blue do not effect growth. * indicates partial dominant negative effect.

94

log10 O.D

10

Wt
K114E
Y225A

1

0.1

0.01

0

50

100

Time in mins

150

200

Figure 3.5 An example of growth curves comparing wild type growth with a mutation that
causes a mild growth defect and a mutation that causes a severe growth defect.
Strains were grown in LB + 2% Xylose at 37°C. Strain RB613 is a control strain with wild type
rbgA gene under the control of Pxyl promoter. In the other strains mutated rbgA gene is under
control of Pxyl promoter. Represented here are examples of mutation that causes a mild growth
defect (K114E) and a mutation that causes a severe growth defect (Y225A).

95

Figure 3.6 In vitro binding assay for RbgA mutants to 50S subunit under different nucleotide
conditions.
Purified RbgA-his6 or RbgA mutant-his6 (50 nM) were incubated with purified 50S (10 nM)
intermediates and GDP, GTP or GMP-PNP (400 µM) at 37°C. The presence of RbgA was tested
by western blot with custom RbgA-antibody and quantified. Percentages were generated by
assigning RbgA + 50S + GMP-PNP as 100%. Lane1: RbgA + 50S + GDP; lane2: RbgA + 50S +
GTP; lane3: RbgA + 50S + GMPPNP; lane4: A206D + 50S + GDP; lane5: A206D + 50S +
GTP; lane6: A206D + 50S + GMPPNP; lane7: Y225A + 50S + GDP; lane8: Y225A + 50S +
GTP and lane 9: Y225A + 50S + GMPPNP.

96

Figure 3.7 A ligand based structural superimposition of EF-Tu over the free RbgA.
Free EF-Tu is depicted in magenta, ribosome bound EF-Tu is depicted in light grey and free
RbgA is depicted in blue color. For clarity only the helices containing the catalytic histidine
residues are shown along with GTP analogue.

97

Figure 3.8 A ligand-based superimposition of RbgA and EF-Tu for comparison of catalytic
machinery is shown.
RbgA G domain is shown in wire form and only switch II and following helix is shown from the
EF-Tu structure (orange). Switch II in EF-Tu harbors catalytic residue His85 while in RbgA it
connects the ANTAR domain (light brown) via the linker helix (light blue). Putative catalytic
residue His9 in RbgA is relocated to a loop connected to a helix near the N terminus (cyan)
which occupies a similar position to the EF-Tu helix connected to His85. Switch II in RbgA
would prevent the direct involvement of His9 from the helix.

98

Figure 3.9 Ribosome profiles of RbgA mutants.
Strain RB301 was grown in the presence of IPTG (wild type) and in the absence of IPTG
(depleting RbgA protein condition). The strains with mutated rbgA were grown in the presence
of xylose to induce the RbgA mutant protein. Ribosome profiles were generated by
centrifugation of lysates through sucrose density gradients (10-25%). Profiles are shown from
the bottom of the gradient (left, 25%) to the top of the gradient (right, 10%). Dashed lines
indicate where 70S, 45S and 30S complexes migrate in the gradients.
99

Figure 3.10 Proteomic analysis of ribosomal intermediate formed in strains expressing RbgA
mutant protein.
45S complex was isolated from strains expressing mutated RbgA protein and from RbgA
depleted strain. The 50S complex was isolated from RB301 grown in the presence of IPTG. The
complexes were run on 12% SDS-PAGE gel and stained with Coomassie blue stain. The
ribosomal intermediates formed in cells expressing mutant RbgA protein is similar in
composition to the 45S intermediate formed in the absence of RbgA. All 45S complexes
analyzed lacked ribosomal protein L16 seen in the 50S subunit – identified using mass
spectrometry.

100

ACKNOWLEDGEMENTS
We thank David Achila and Brian Shy for technical assistance.

101

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102

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106

CHAPTER 4
Functional interaction between ribosomal protein L6 and RbgA during ribosome assembly

Part of the research presented in this chapter was
performed by Nikhil Jain, who contributed
Figures 4.11, 4.12 and 4.13

107

Abstract
RbgA is an essential GTPase that participates in the assembly of the large ribosomal subunit in
Bacillus subtilis and its homologs are implicated in mitochondrial and eukaryotic large subunit
assembly. How RbgA functions in this process is still poorly understood. To gain insight into the
function of RbgA we isolated suppressor mutations that partially restored the growth of an RbgA
mutation (RbgA-F6A) that caused a severe growth defect. Analysis of these suppressors
identified mutations in rplF, encoding ribosomal protein L6. The suppressor strains all
accumulated a novel ribosome intermediate that migrates at 44S in sucrose gradients. All of the
mutations cluster in a region of L6 that is in close contact with helix 97 of the 23S rRNA. In
vitro maturation assays indicate that the L6 substitutions allow the defective RbgA-F6A protein
to function more effectively in ribosome maturation. Our results suggest that RbgA functions to
properly position L6 on the ribosome, prior to the incorporation of L16 and other late assembly
proteins.

108

Introduction
The assembly of the 30S and 50S ribosomal subunits is a complex and tightly coordinated series
of events that consists of the synthesis, processing and modification of 5S, 16S and 23S rRNA
and the addition of more than 50 ribosomal proteins (r-proteins) [1-3]. The in vitro reconstitution
of a mature 50S subunit has been extensively studied in Escherichia coli and the formation of a
mature 50S subunit from its constituent r-proteins and rRNA is a multi-step process that requires
non-physiological conditions such as high ionic concentration, high temperatures and long
incubation times [4-7]. Relatively fewer studies focused on ribosome assembly in other bacterial
species, such as Bacillus stearothermophilus, and these demonstrated that the intermediates
formed in this system are different than those in E. coli, however similar non-physiological steps
are required for formation of a functional ribosomal subunit [5,8]. Moreover, recent studies have
utilized biophysical techniques to study ribosome assembly in vivo and demonstrated that
assembly of the ribosome subunits is a multistage process that appears to follow multiple parallel
pathways in which the accumulation of assembly intermediates identified in vitro do not
accumulate in vivo [9-11]. The slow kinetics and attenuated efficiency of in vitro assembly
strongly suggest that assembly factors are involved in vivo and indeed, several classes of
assembly factors such as GTPases, RNA helicases, RNA modification enzymes and chaperone
proteins have been implicated in in vivo ribosome assembly in bacterial and eukaryotic cells
[2,12-15]. However, while studies show that these factors are functionally significant and play a
critical role in ribosome assembly, the molecular functions of these factors remain elusive. RbgA
(ribosome biogenesis GTPaseA) is an essential GTPase that is required for late step assembly of
the 50S subunit in Bacillus subtilis [16,17]. RbgA is a widely conserved protein and its
eukaryotic homologs such as Mtg1, Lsg1, Nug1 and Nog2 have also been implicated in assembly

109

of the large ribosomal subunit [18-21]. RbgA depleted cells do not form mature 50S subunits but
instead accumulate a 45S complex. Quantitative mass spectrometry analysis of this particle
shows that the 45S completely lack ribosomal proteins L16, L28, L36 and contain severely
reduced amount of L27, L33a and L35 [16,22]. Proteins L16 and L27 are crucial components of
the peptidyltransferase center in 50S subunit and directly contact the A-site and the P-site
respectively [23,24]. Functional studies have shown that both proteins play a role in stabilization
of the peptide bond formation, the positioning of tRNA on their respective sites and are required
for optimal functioning of the ribosome [25-27]. While there have been no reports of deletion of
L16, the deletion of L27 in E. coli causes a severe growth defect [28]. However, studies in B.
subtilis indicate that both proteins are essential and deletion mutants could not be obtained for
either protein [29]. In vitro assembly experiments have demonstrated that incorporation of L16
into the growing complex occurs at a late stage in the assembly process and is accompanied by a
large conformational change [30]. In yeast, the RbgA homolog Lsg1 has been proposed to play a
role in the incorporation of the L16 homolog Rpl10 into the large ribosomal subunit, suggesting
that RbgA and its homologs regulate an evolutionarily conserved step during biogenesis [31,32].
RbgA has been shown to interact directly with both the 45S complex and the 50S subunits and
the GTPase activity of RbgA is enhanced ~60 fold in the presence of the mature 50S subunit
[33]. Mutational analysis of RbgA has shown that a stretch of 15 amino acids in the N-terminal
domain, which is largely conserved among all bacterial RbgA homologs as well as eukaryotic
homologs, plays a crucial role in GTPase activity [34]. Mutations that affect GTP hydrolysis
result in the accumulation of the 45S complex similar to RbgA depleted cells indicating that GTP
hydrolysis plays a key role in maturation of the 50S subunit [34].

110

To further investigate the role of RbgA in the assembly of the 50S subunit we constructed a B.
subtilis strain that expressed a mutated RbgA protein that results in a severe growth defect and
screened for suppressors that alleviated this growth defect. We isolated and characterized eight
independent suppressor strains and found they contained six distinct mutations in the rplF gene,
which encodes for ribosomal protein L6. Analysis of ribosome assembly in these strains led to
discovery of a novel ribosomal intermediate that differs from the 45S complex observed in the
parental strain and also in RbgA-depleted cells. We discuss the implications of these results and
present a possible model for the role of RbgA in assembly of the 50S subunit.

Materials and Methods
Growth conditions
All strains were grown at 37°C in LB medium and cultures were shaken at 250 rpm. Antibiotics
were added at the following concentrations when required: chloramphenicol (5µg/ml),
erythromycin (5µg/ml), lincomycin (12.5µg/ml), spectinomycin (100µg/ml) and ampicillin
(100µgml). IPTG was added to a final concentration of 1mM when required for strain growth.

Plasmids
Plasmid pMA1 was derived from pSWEET, an amyE insertion vector with a chloramphenicol
resistance cassette, by placing the rbgA gene under the control of a xylose inducible promoter.
Plasmid pAS24, an amyE insertion vector with a spectinomycin resistance, was used to construct
pMG28 by inserting a wild-type copy of rbgA under the control of its native promoter. Plasmid
pMG29 bearing a F6A mutation in the rbgA gene (accomplished by a TTC to GCC codon
change) was constructed from pMG28 using the QuikChange II XL kit (Stratagene) by following

111

the manufacturer’s instructions. Plamid pJCL87 was derived from pDR111 and contains a
chloramphenicol resistance cassette and the IPTG inducible Phyperspankpromoter. Plasmid
pMG30 was constructed from pJCL87 by cloning the first 330 bp of the map gene under the
control of the Phyperspankpromoter.

Construction of strains
All strains used in this study are derived from the wild type strain JH642 (RB247) and listed in
Table 4.1. The construction of strain RB301 and RB418 has been described previously [16].
RB395 was constructed by transforming RB247 with pMA1 and knocking out the native rbgA
gene by using a MLS cassette. Strain RB1006 was constructed by transforming RB247 with
plasmid pMG28 at the amyE locus and knocking out the native rbgA gene by using a MLS
cassette. The strains were checked for interruption of amyE by growth on starch plates. Strain
RB1043 was constructed by transforming RB247 with plasmid pMG29 and knocking out the
native rbgA gene by using chromosomal DNA from RB395. Independently, strain RB1044 was
constructed in a manner identical to RB1043 to serve as a biological duplicate. All strains
discussed in this study were confirmed for desired change using PCR to amplify the region of
interest followed by sequencing.

Suppressor screen
Strains RB1043 and RB1044 were used for suppressor analysis. A single colony from each of
these strains was inoculated per flask (25 colonies per strain, total of 50 colonies) and grown at
37°C for 16 hours. The undiluted culture from each flask as well as two serial dilutions (10-, and
100-fold) were plated on LB plates and incubated overnight at 37°C. The parental strains
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RB1043 and RB1044 were also plated along with RB1006 carrying wild-type RbgA to serve as
controls. Isolated colonies from eight strains-RB1051, RB1055, RB1057, RB1059 (from
RB1043) and RB1061, RB1063, RB1065, and RB1068 (from RB1044) that grew faster than
parental strains were identified and characterized further.

Whole genome sequencing and bioinformatics analysis
Genomic DNA from RB247, RB1043, RB1051, RB1055, RB1057, RB1059, RB1061, RB1063,
RB1065 and RB1068 was isolated using the Wizard genomic DNA isolation kit (Promega). The
genomic DNA was analyzed on a 0.8% agarose gel to ensure that the quality was suitable for
sequencing. Whole genome sequencing was performed on a Genome Analyzer II instrument
equipped with a paired end module (Illumina) at the MSU RTSF. The sequencing reads obtained
were quality tested using FASTQC and trimmed if needed. Next we aligned sequence reads from
RB247 and RB1043 against the reference B. subtilis strain 168 genome using R2R software. We
identified the insertion of pMG29 in RB1043 when compared with RB247 reads and the
insertion of the MLS cassette in RB1043 at the native rbgA locus. The sequence of suppressor
strains RB1051, RB1055, RB1057, RB1059, RB1063, RB1065 and RB1068 was then compared
to RB1043 (the parental strain). In addition to the expected insertions found in RB1043 and each
suppressor strain (corresponding to pMG28 at the amyE locus and the MLS cassette at the native
rbgA locus) we identified only a single change in each suppressor strain in the rplF gene. The
suppressor mutations that were identified utilizing the R2R platform were confirmed by PCR
amplification of the rplF gene and sequencing the amplified product.

113

Structure analysis
Homology model of L6 from B. subtilis was obtained by using Modeller 9.12 [35], utilizing the
crystal structure of L6 (PDB code: 1RL6) from B. stearothermophilus as a template. Out of 20
models constructed, the model with lowest energy (molpdf) was chosen for further analysis. All
structural analysis for figure 7 were carried out in Chimera using the 50S structure (PDB:
2AW4) [35,36].

Constructions of strains for determining the phenotype of the L6 protein in a wild-type
background
Strain RB1095 was constructed by transforming RB247 with pMG30 such that that the
expression of the map gene (at the end of the operon that contains the rplF gene) was controlled
by the IPTG inducible Phyperspank promoter. RB1102 was constructed by transforming
suppressor strain RB1051 with chromosomal DNA from RB1095 and selecting cells on IPTG,
chloramphenicol and MLS (lincomycin and erythromycin) such that the rbgA-F6A gene at amyE
locus was selected and the mutated rplF gene operon was tagged with chloramphenicol marker.
RB1103, RB1106 and RB1107 were constructed similarly by using RB1055, RB1065 and
RB1068 as the parental strains, respectively. RB1117 was constructed by transforming RB247
with chromosomal DNA from RB1102 and selecting cells on IPTG and chloramphenicol, thus
ensuring that this strain had wild type rbgA gene at the native locus and the mutated rplF gene
(operon was tagged with the chloramphenicol marker). RB1118, RB1121 and RB1122 were
constructed similarly by utilizing chromosomal DNA from RB1103, RB1106 and RB1107
respectively. RB1123 was constructed by growing RB1117 on LB plates without
chloramphenicol and IPTG such that the plasmid pMG30 was excised out leaving the mutated

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rplF gene in a wild type background. RB1125, RB1131and RB1133 were constructed similarly
from RB1118, RB1121 and RB1122 respectively.

Analysis of ribosome profiles and ribosome complexes
Ribosomal subunits were prepared by sucrose density centrifugation. 50S and 45S complexes
were isolated from lysates of RB418 and RB301 cells, respectively as previously described [34].
RB1051, RB1055, RB1057, RB1063, RB1065 and RB1068 were grown to OD600 of 0.5 at 37°C
in LB medium. Chloramphenicol (Sigma) was added to a final concentration of 100μg/ml 5
minutes prior to harvesting. Cells were harvested by centrifugation at 5000g for 10 min and
resuspended in lysis buffer [10 mMTris-HCl (pH 7.5), 60 mMKCl, 10 mM MgCl2, 0.5% Tween
20, 1 mM DTT, 1x Complete EDTA-free protease inhibitors (Roche) and 10 U/ml RNase-free
DNase (Roche)]. Cells were lysed by three consecutive passes through a French press set at 1400
to 1600 psi and clarified by centrifugation at 16000xg for 20 minutes. Clarified cell lysates were
loaded on top of 10-25% sucrose density gradients equilibrated in buffer B (10 mMTris-HCl, pH
7.6, 10 mM MgCl2, 50 mM NH4Cl) and centrifuged using a SureSpin 630 rotor (Sorvall) for 4.5
hours at 30,000 rpm. Gradients were then fractionated on a BioLogic LP chromatography system
(BioRad) by monitoring UV absorbance at 254 nm. Fractions corresponding to ribosomal
subunits of interest were pooled, concentrated using 100kDa cutoff filters (Millipore) and stored
in buffer A (10 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 60 mM KCl and 1 mM DTT) at -80°C.
Ribosomal subunit peak fractions were pooled, concentrated, and stored in buffer A (10 mMTrisHCl, pH 7.6, 10 mM MgCl2, 60 mMKCl and 1 mM DTT) at -80°C. To visualize L16 in the
subunits/intermediates 5 pmoles of each subunit to be analyzed was run on 12% NuPAGE SDS-

115

PAGE gel (Invitrogen) at 120V in MOPS buffer (Invitrogen) and stained using SimplyBlue
SafeStain (Invitrogen) following the manufacturer’s instructions. To visualize L6 protein in the
subunits/intermediates 10 pmol was loaded on a 28cm long 15% Bis-Tris gel and stained using
Coomassie blue stain. L6 protein was identified by LC/MS/MS. To visualize L6 on a mini gel 5
pmol of subunits were loaded on a 16% Tricine gel (Invitrogen) and stained using SimplyBlue
SafeStain.

In vitro maturation
Cell lysates from RB1043 and RB1055 were obtained as described above. Lysates were
concentrated using 4mL Amicon ultra-4 centrifugal filters with 4 kda cutoff (Millipore). An
equal volume of lysate was incubated at 37oC or 0oC for 1 hour then loaded onto 18-43% sucrose
gradient made in buffer C (10 mMTris-HCl, pH 7.6, 20 mM MgCl2, 50 mM NH4Cl) followed by
centrifugation at ~82000 g for 14 hours at 4oC in Surespin 630 rotor (Sorvall). Gradients were
fractionated on BioLogic LP system (BioRad) monitoring absorbance at 254 nm.

GTPase activity
The assay was performed as described [34]. Briefly, for measuring GTPase activity in the
presence of ribosomal subunits/intermediates 100nM RbgA protein was incubated with 100nM
50S subunit or 45S subunit or 44S subunit and 200µM GTP at 37°C for 30 minutes and for
measuring intrinsic GTPase activity 2µM RbgA protein was incubated with 200µM GTP at 37°C
for 30 minutes. We predetermined that under these conditions the values were in the linear range
of the assay. The phosphate released was measured using the Malachite Green Phosphate Assay
Kit (BioAssaySystems).

116

Results
Construction of a strain containing a rbgA mutation with a growth phenotype suitable for a
genetic suppressor screen
To generate a strain that displayed a strong growth defect that would be amenable to suppressor
analysis, we analyzed the phenotypes of over 40 site-directed mutations in the rbgA gene [34].
We were interested in identifying substitutions in RbgA that displayed reduced GTPase activity
upon association with the ribosome and were still able to bind to the ribosome. One such
mutation, rbgA-F6A, was identified as meeting both of these criteria. Our results showed that
GTPase activity of RbgA-F6A was reduced ~12 fold, however the mutation did not prevent
stable association with the 45S complex and the 50S subunit [34]. Therefore we constructed a
strain in which rbgA-F6A was the only functional copy of rbgA in the cell expecting that cells
harboring rbgA-F6A would be viable but display reduced growth. To achieve this we constructed
strain RB1043 by cloning the rbgA gene (containing a mutation that results in a F6A
substitution) fused to its native promoter into the plasmid pAS24 and inserted this construct at
the amyE locus (Table 4.1). A control strain (RB1006) that contains a wild-type copy of the rbgA
gene at the amyE locus was also constructed in similar manner as a control. The native rbgA
gene was inactivated in both strains by the insertion of a MLS cassette by marker replacement,
which led to the complete removal of the rbgA gene. Comparison of the two strains showed that
the strain expressing RbgA-F6A (RB1043) was severely growth compromised and exhibited a
growth rate~7 fold slower than the RB1006 strain (Figure 4.1A). This severe growth defect was
utilized to isolate suppressors that restored the ability of this strain to grow more rapidly.

117

To isolate independent, spontaneous suppressor mutations we inoculated a single colony of the
RB1043 (rbgA-F6A) strain per flask into a total of 50 flasks and isolated suppressors that
exhibited faster growth at 37°C (only one per flask). We identified eight independent suppressor
strains that partially alleviated the growth defect of RB1043 (Figure 4.1 and Table 4.2).
Individual suppressors were grown in liquid medium and their growth rates were compared to
the parental RB1043 strain and the control strain RB1006. The wild-type control strain RB1006
and the parental RB1043 strains exhibited a doubling time of 23 minutes and 173 minutes,
respectively, whereas the growth rate of the suppressor strains ranged from 46 to 77 minutes
(Table 4.2). Next, we sequenced the rbgA-F6A gene to check for reversion mutations and found
that all eight strains did not contain any intragenic suppressor mutations. We then proceeded to
backcross each suppressor strain with the wild-type RB247 strain and inactivated the native rbgA
gene. The reappearance of RB1043 phenotype (~ 7-fold increase in doubling time) in each
backcrossed strain indicated that the suppressor mutation was unlinked to the rbgA-F6A
mutation.

Suppressor mutations localize to the rplF gene, which encodes the ribosomal protein L6
To identify the genetic changes responsible for the partial suppression of the growth defect we
obtained the whole genome sequence of all eight suppressor strains, RB247 (wild-type
background) and the parental RB1043. The resultant sequence reads were mapped back to the B.
subtilis reference genome. The sequence reads from parental RB1043 strain were compared with
each suppressor strain sequentially. After accounting for mutations that have arisen in our
genetic background or were sequencing errors in the original B. subtilis sequencing project [37],
the analysis revealed that each suppressor strain had a single point mutation compared to the

118

RB1043. In each case a single point mutation in the rplF gene, encoding ribosomal protein L6,
was found. Three suppressor strains had the same mutation (Table 4.2) and thus we obtained six
unique suppressor mutations that caused single amino acid substitutions in L6; R3C, G5C, G5S,
H66L (3 isolates), T68R and R70P. Alignment of L6 proteins from phylogenitcally diverse
bacteria indicates that these residues are conserved in bacterial L6 proteins, with T68
demonstrating the most conservation when compared to L6 homologs from archaea and
eukaryotes (Figure 4.2). We constructed a homology model of the B. subtilis L6 protein based on
the structure of the L6 protein from B. stearothermophilus and mapped the suppressor mutations
onto the modeled structure of the protein. Our analysis shows that all of the six suppressor
substitutions reside in close vicinity in the protein structure (Figure 4.3) and are contained within
the N-terminal structural domain.

Suppressor strains accumulate a novel ribosomal intermediate that is distinct from the 45S
particle
To assess the status of ribosome assembly in the suppressor strains, we analyzed the ribosome
profiles using 10-25% sucrose density gradients. Our results showed that all of the suppressor
strains accumulated a novel ribosomal intermediate that migrated at ~44S and was distinct from
the 45S complex that accumulates in RbgA-depleted cells and RB1043 strain expressing RbgAF6A (Figure 4.4).
We isolated the 44S intermediates from each suppressor strain and analyzed the protein
composition utilizing SDS-PAGE (Figure 4.5 and 4.6). The 44S intermediates from RB1055
(L6-R3C) and RB1065 (L6-G5S) exhibited significantly reduced levels of L6 whereas RB1057
(L6-H66L) and RB1063 (L6-G5C) showed a moderate reduction (Figure 4.5 and 4.6). Strains

119

RB1051 (L6-R70P) and RB1068 (L6-T68R) accumulated 44S intermediates with L6 protein
levels similar to that observed for the 45S complex and the 50S mature subunit (Figure 4.6 and
4.6). Most of the 44S subunits lacked detectable L16, however we noticed that one 44S
intermediate [RB1068 (T68R)] stably incorporated L16 to near wild-type levels (Figure 4.5 and
4.6). It is important to note that while the protein composition of the 44S intermediate with
regard to the levels of L6 and L16 differs among the suppressor strains, the migration of this
complex in each suppressor strain does not change, indicating that the difference in migration is
likely due to additional conformational changes and/or other missing ribosomal proteins.
Next we analyzed the GTPase activity of RbgA in the presence of the 44S intermediate that
accumulate in the suppressor strains. We isolated 44S particles from each suppressor strain to
test if the levels of L6 or L16 in the complex influence the GTPase activity of RbgA. Our results
show that GTPase activity of RbgA is stimulated ~4-6 fold in the presence of the 44S
intermediates (Figure 4.7). This is similar to the fold change in GTPase activity in the presence
of the 45S complex and highly reduced compared with the ~60 fold stimulation seen in the
presence of the mature 50S subunit [33].

rplF mutations do not impair growth but are partially defective in ribosome subunit
joining
We were interested in studying the effects of the alterations in ribosomal protein L6 on cell
growth and ribosome assembly in an otherwise wild-type background. To do this we created
strains in which the rplF mutations were linked to an antibiotic resistance marker and moved into
a wild-type background RB247. . Once each mutation was transferred to a wild-type background,
the antibiotic resistance marker was easily removed by passage on media without selection,

120

resulting in strains that only contained mutations in rplF (see materials and methods for details).
We successfully constructed strains in which mutations in rplF resulting in the R3C (RB1125),
G5S (RB1131), T68R (RB1133), and R70P (RB1123) substitutions were the only alterations in
the chromosome (Table 4.1). Each L6 mutant strain grew at a doubling time that was
indistinguishable from the congenic wild-type RB247 strain, demonstrating that the partial
suppression of the RbgA-F6A growth phenotype was not due to an impairment of growth due to
defects in L6.
Although the rplF mutations did not have an effect on cell growth, we were interested to identify
if they had any impact on ribosome maturation. Ribosome profiling of strains RB1123, RB1125,
RB1131 and RB1133 through 10-25% sucrose gradients was performed and in each case L6
substitutions resulted in abnormal ribosome profiles. Figure 4.8 depicts the ribosome profile of
RB1123 (L6-R70P) and demonstrates that despite displaying a wild-type growth rate, the mutant
has an increased level of individual ribosomal subunits when compared to wild-type cells. Each
of the other three mutants tested also had altered ribosome profiles that were similar to RB1123,
indicating that the L6 substitutions impact subunit joining or maintaining 70S ribosome stability
(Figure 4.9). We further analyzed the 50S subunits that accumulated in these strains and found
that both ribosomal proteins L6 and L16 were present in levels similar to wild-type 50S subunits
(Figure 4.10) indicating that these substitutions in RplF (R3C, G5S, T68R and R70P) do not
impact the association of L6 or L16 with the 50S subunit.

The 44S particle can be matured into a 50S subunit in vitro
One possible mechanism for how L6 substitutions may suppress the RbgA-F6A defect is that
44S particles may be more easily matured into 50S subunits than 45S particles. To address this

121

possibility we concentrated purified lysates from RB1043 (RbgA-F6A) and RB1055 (RbgAF6A, L6-R3C) and incubated them at either 37°C and 0°C for 1 hour. After incubation, these
2+

lysates were centrifuged over 18-43% sucrose gradients in the presence of 20mM Mg

(to

facilitate mature subunit joining since L6 mutants show subunit association defects in 10mM
2+

Mg , see Figure 4.8). Concentration of lysates (see materials and methods) led to an increase in
70S formation in both of the RB1043 and RB1055 lysates (data not shown). Incubation of the
RB1055 lysate at 37°C demonstrated that many of the 44S particles in the RB1055 lysate were
converted into 50S subunits that subsequently partnered with the 30S subunits to form 70S
ribosomes (Figure 4.11). 70S ribosomes showed a more than 100% increase during 37°C
incubation with a concomitant decrease in 44S and 30S subunits (Figure 4.11). These data
support the idea that 44S particles are able to be matured more quickly into 50S subunits than
45S particles.

Discussion
We provide evidence that mutations causing substitutions in the N-terminal domain of ribosomal
protein L6 can suppress ribosome assembly defects associated with a mutation that impairs the
function of the ribosome assembly factor RbgA. The suppressor substitutions in L6, when
coupled with the RbgA-F6A defect, gave rise to novel 44S particles. We propose that the partial
suppression of growth and ribosome assembly defects observed are not due to the fact that the L6
substitutions bypass the requirement for RbgA in the cell. This is supported by two observations.
First, we have repeatedly attempted to generate an rbgA null mutation in the background of
several of the rplF suppressor mutations but were unsuccessful. Second, the fact that L6
substitutions do not cause a growth defect or accumulate a 44S particle in a wild-type
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background supports that the function of the RbgA-F6A protein is still required for maturation.
We suggest that the L6 alterations allow the defective RbgA-F6A protein to function more
effectively in ribosome assembly. The in vitro maturation assay further supports this idea, as
evidenced by increased maturation of the 44S particle observed in RB1055 (RbgA-F6A, RplFR3C) versus the RB1043 (RbgA-F6A) strain. In addition, all of the L6 supressors lead to
increased 70S ribosome formation in vivo. Since the only copy of RbgA in both strains carries
the F6A substitution, this indicates that the L6 substitutions, along with the partial function of
RbgA, are required to facilitate large subunit maturation.
L6 is a two-domain protein that is located on the L7/L12 side of the 50S subunit and forms an Llike structure that appears to form a clamp from the front to the back of the subunit (Figure 4.12)
[38,39]. The N-terminus of the protein interacts with helix 97 (h97) of 23S rRNA, while the Cterminus of L6 interacts with the sarcin/ricin loop (SRL) [38,40,41]. All of the L6 substitutions
that suppress the RbgA-F6A defect map to a small region in the N-terminus of the protein and in
some cases disrupt direct interactions between L6 and h97 (Figure 4.12). Although some of the
suppressor mutations cause L6 to unstably associate with the 44S intermediate, the ability to
suppress the RbgA-F6A defect does not seem to correlate with L6 binding as two of the 44S
particles isolated from the suppressors (R70P, H66L) have near wild-type levels of L6. However,
the conformation of mutated L6 might be different in 44S as the structures of the ribosome
intermediates that form in the suppressor strains are clearly altered based on their slower
migration within sucrose gradients. The consequence of the L6 substitutions in a wild-type
background appears to be at the level of 70S stability. Individual 50S subunits that contain
mutant L6 proteins appear to have normal amounts of both L6 and L16, indicating that once
matured these proteins are stably incorporated. However, clearly there is some disruption of 50S

123

subunit structure that causes decreased stability of 70S ribosomes. This is possibly due to
improper positioning of the intersubunit bridge helix 89, which is located between and makes
direct contacts with L16 and L6.
What effect might mutations in L6 have in suppressing ribosome assembly defects associated
with reduced function of RbgA? L6 binds prior to L16 and has been implicated in setting up the
binding site for L16 [4,42]. In E. coli, the expression of ribosomes that are deleted for the SRL,
which interacts with the C-terminus of L6, are dominant-lethal and result in the accumulation of
50S subunits that lack L16 [43]. Lancaster et al. propose that L6 binds to the assembling subunit
via initial interactions between the N-terminus of L6 and h97, which then results in the
subsequent assembly of the functional core of the 50S subunit [43]. This includes the formation
of several key interactions between h97, h42, h89, h91, and h95, which are predicted to be
initiated by the initial binding of L6 with h97. When the SRL is deleted these interactions are
disrupted and the L16 binding site, along with other functional regions of the large subunit, are
improperly assembled and non-functional. The L6-T68R mutant may therefore be an interesting
mutant to study as it contains not only near normal levels of L6 and substantial levels of L16,
despite still existing as a 44S particle.
Although we still do not know the precise role that RbgA plays during ribosome assembly, the
identification of the second-site suppressors in L6 supports a model in which RbgA participates
in facilitating the correct association of L6 with the ribosome to allow subsequent maturation
events to take place (Figure 4.13). Recent studies have postulated that ribosomal subunits can be
formed via multiple parallel pathways. We suspect that the large subunit pathways converge on a
late assembly intermediate (LAI50-1) and GTPases, such as RbgA, act on LAI50-1 to complete
maturation. We envision two scenarios in which RbgA could act on LAI50-1 to facilitate

124

maturation. In scenario 1, RbgA binds to an undefined late assembly intermediate (LAI50-2), and
promotes the rearrangement or movement of helix 97 to facilitate the correct incorporation of L6.
In scenario 2, L6 binds to the ribosome prior to RbgA (resulting LAI50-3, equivalent to the 45S
complex) in an unproductive interaction 275 and the role of RbgA binding is to promote the
correct interaction of L6 with the helix 97 [43]. Recently, we have shown that the 45S particle is
not a dead end particle and can be fully matured into a 50S particle in vivo [22]. The fact that L6
is not fully visible in the cryo-EM structures of the 45S complex provides support that L6 is not
in its proper conformation [22]. In both scenarios, correct positioning of L6 and h97 allows for
proteins L16, L27, L28, L33, L35, and L36 to be stably incorporated into the large subunit. Once
RbgA senses that incorporation of these proteins has taken place GTP hydrolysis occurs, a final
maturation event takes place, and RbgA leaves the subunit. Because we have not been able to
isolate RbgA mutants that are deficient in GTPase activity that form 50S subunits, we predict
that the GTP hydrolysis plays a dual role in both promoting conformational changes in the
ribosome while also resulting in RbgA leaving the subunit. Support for this latter step stems from
the fact that 50S subunits lacking only ribosomal proteins L16 and L28 do not stimulate the
GTPase activity to levels observed with wild-type 50S subunits [22].
Although we do not know the order of binding of L6 and RbgA, in both scenarios the proposed
role of RbgA is to properly position L6 and helix 97 to facilitate assembly. This interaction
between L6 and h97 is evolutionarily conserved (see Figure 4.14) and, given that RbgA
homologs are present in archaea and eukaryotes, the role of RbgA proteins in ribosome assembly
is likely to be conserved as well. Thus it appears that in small subunit and large subunit ribosome
biogenesis, one function of assembly factors is to prevent binding of late binding ribosomal
proteins until the subunit is ready to receive them [22,44,45]. Whether or not these potential
125

checkpoints are related to quality control mechanisms that insure only functional ribosomes enter
into translation remains to be seen [45]. Interestingly, E. coli and many other proteobacteria lack
RbgA, a function that was present in the last common ancestor and subsequently lost in this
lineage of bacteria. We are currently using a comparative genomics approach to identify
differences between E. coli and B. subtilis ribosomes in an attempt to further localize the precise
site and mechanism of RbgA function.

126

Strain

Relevant genotype

Source

RB301

JH 642 Pspank-rbgAcat pMAP65

[16]

RB419

JH 642 Pspank-infBcat pMAP65

[16]

RB1006 JH 642 ΔrbgA::MLS amyE::rbgASpcr

This study

RB1032 JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpcr

This study

RB1043 JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpcr

This study

RB1044 JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpcr

This study

RB1051 JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpcrrplF-R70P

This study

RB1055 JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpcrrplF-R3C

This study

RB1057 JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpcrrplF-H66L

This study

RB1059 JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpcrrplF-H66L

This study

RB1061 JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpcrrplF-H66L

This study

RB1063 JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpcrrplF-G5C

This study

JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpcrrplF-G5S

This study

Rb1065

r

RB1068 JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpc rplF-T68R

This study

RB1095 JH 642 adk::Cm Pspank map

This study

RB1102
RB1103
RB1106
RB1107

JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpcrrplF-R70P adk::Cm Pspank
map
JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpcrrplF-R3C adk::Cm Pspank
map
JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpcrrplF-G5S adk::Cm Pspank
map
JH 642 ΔrbgA::MLS amyE::rbgA-F6ASpcrrplF-T68R adk::Cm Pspank
map

This study
This study
This study
This study

RB1117 rplF-R70P adk::Cm Pspank map

This study

RB1118 rplF-R3C adk::Cm Pspank map

This study

RB1121 rplF-G5S adk::Cm Pspank map

This study

RB1122 rplF-T68R adk::Cm Pspank map

This study

RB1123 JH 642 rplF-R70P

This study

Table 4.1 List of strains used in this study
127

Table 4.1 (cont’d)
RB1125 JH 642 rplF-R3C

This study

RB1131 JH 642 rplF-G5S

This study

RB1133 JH 642 rplF-T68R

This study

128

Suppressor/Strain

Strain number

rplF gene

L6 protein

codon
change

Amino acid
change

Doubling
time
(mins)

Control strain

RB1006

Wild type

none

23 ± 1

Suppressor 1

RB1055

cgt to tgt

R3C

69 ± 1

Suppressor 2

RB1063

ggt to tgt

G5C

77 ± 1

Suppressor 3

RB1065

ggt to agt

G5S

52 ± 10

Suppressor 4, 5 and 6

RB1057, RB1059,
RB1061

cat to ctt

H66L

66 ± 10

Suppressor 7

RB1068

acg to agg

T68R

49 ± 5

Suppressor 8

RB1051

cgc to ccc

R70P

46 ± 4

Parental strain (RbgAF6A)

RB1043

Wild type

none

173 ± 5

Table 4.2 Suppressor mutations in ribosomal protein L6
Values represent three independent experiments ± S.D.

129

Figure 4.1 Phenotype of RB1043 (rbgA-F6A) and suppressor strains.
A. RbgA-F6A mutation causes a severe growth defect. Strains were grown in LB at 37°C. Strain
RB1006, depicted by closed circles, is a control strain with wild type rbgA gene under the
control of native promoter at amyE locus. RB1043, depicted by closed triangles, expresses rbgAF6A gene under the control of native promoter at amyE locus. B. RB1006 (Wild-type control
strain), C.RB1043 and RB1044. RB1044 is an independent isolate that contains the rplF-F6A
mutation and has an identical phenotype. D. Two suppressor strains isolated from RB1043,
(RB1051 and RB1068), that partially alleviate the growth defect. All strains were cultured on LB
plates at 37°C overnight.

130

A.

Figure 4.2 Multiple sequence alignment of L6 protein from selected bacterial species.
Substitutions in rplF partially suppress the growth defect of rbgA-F6A. The positions of the
mutated residues are highlighted. Alignments were constructed with ClustalW with default
parameters and species indicated to the left. ‘*’ indicates positions which have a single, fully
conserved residue, ‘:’ indicates conservation between groups of strongly similar physicochemical
properties, ‘.’ indicates conservation between groups of weakly similar physicochemical
properties.

131

Figure 4.2 (cont’d)
B.

132

Figure 4.2 (cont’d)
C

133

Figure 4.3 Homology model of L6 protein depicting the suppressor mutations.
Homology model of L6 protein of B. subtilis is shown as a surface representation in grey. The
residues that are mutated in the suppressor strains, highlighted in red with the corresponding
amino acid labeled.

134

Figure 4.4 The ribosome profile of RbgA-F6A suppressor strain show an accumulation of a
novel 44S complex.
Ribosome profiles were analyzed from RB1051 (A), RB1055 (B), RB1057 (C), RB1065 (D),
RB1068 (E) and RB1063 (F). The ribosome profile from the suppressor strains were aligned
with ribosome profiles from RbgA depleted cells (panel 2), IF-2 depleted cells (panel 3) and
RB1043 (panel 4). Profiles are generated from the bottom of the gradient (25%) to the top of the
gradient (10%) by ultracentrifugation for 16 hours. (G-H) Ribosome profiles were analyzed
through a 10-25% sucrose density gradient after ultracentrifugation for 3.5 hours. All suppressor
strains accumulated a 44S complex that differs in migration through the gradient from the 45S
complex seen in the profile of RbgA depleted cells and parental strain RB1043 that expresses
RbgA-F6A and the 50S ribosomal subunit seen in IF-2 depleted cells. Profiles are generated
from the bottom of the gradient (25%) to the top of the gradient (10%). Dashed lines indicate the
migration of the 45S and the 30S complex in the gradient.

135

Figure 4.4 (cont’d)

136

Figure 4.4 (cont’d)

137

Figure 4.4 (cont’d)

138

Figure 4.5 Analysis of ribosomal proteins in 44S intermediates.
A. Ribosomal protein L6 is not stably incorporated in all 44S intermediates isolated from the
suppressor strains. Lane 1: 45S isolated from RB301 by depletion of RbgA; lane 2: 45S complex
isolated from RB1043 (rbgA-F6A); lane 3: 44S complex isolated from RB1051 (rplF-R70P);
lane 4: 44S complex isolated from RB1055 (rplF-R3C); lane 5: 44S complex isolated from
RB1057 (rplF-H66L); lane 6: 44S complex isolated from RB1063 (rplF-G5C); lane 7: 44S
complex isolated from RB1065 (rplF-G5S); lane 8: mature 50S subunit isolated from RB418 by
depletion of IF2 and lane 9: BioRad PrecisionPlus AllBlue protein marker. L6 protein was
confirmed by LC/MS/MS. All complexes were analyzed on a 28cm 15% Bis-Tris gel. B.
Ribosomal protein L6 is stably integrated in 44S intermediate isolated from RB1068. Lane 1:
45S isolated from RB301 by depletion of RbgA; lane 2: mature 50S subunit isolated from
RB418 by depletion of IF2; lane 3: 44S complex isolated from RB1055 (rplF-R3C) and lane 4:
44S complex isolated from RB1068 (rplF-T68R). All complexes were analyzed on 16% Tricine
gel. (C) 44S complex isolated from RB1068 contains ribosomal protein L16. Lane 1: 45S
isolated from RB301 by depletion of RbgA; lane 2: mature 50S subunit isolated from RB418 by
depletion of IF2; lane 3: 45S complex isolated from RB1043 (rbgA-F6A); lane 4: 44S complex
isolated from RB1055 (rplF-R3C) and lane 5: 44S complex isolated from RB1068 (rplF-T68R).
All complexes were analyzed on 12% Bis-Tris gel.

139

Figure 4.6 Analysis of ribosomal protein composition of 44S intermediates.
A. Ribosomal protein L6 is not stably incorporated in all 44S intermediates isolated from the
suppressor strains. Lane 1: 45S isolated from RB301 by depletion of RbgA; lane 2: 45S complex
isolated from RB1043 (rbgA-F6A); lane 3: 44S complex isolated from RB1051 (rplF-R70P);
lane 4: 44S complex isolated from RB1055 (rplF-R3C); lane 5: 44S complex isolated from
RB1057 (rplF-H66L); lane 6: 44S complex isolated from RB1063 (rplF-G5C); lane 7: 44S
complex isolated from RB1065 (rplF-G5S); lane 8: mature 50S subunit isolated from RB418 by
depletion of IF2 and lane 9: BioRad Precision Plus All Blue protein marker. L6 protein was
confirmed by LC/MS/MS. All complexes were analyzed on a 28cm 15% Bis-Tris gel.
B. Ribosomal protein L6 is stably integrated in 44S intermediate isolated from RB1063. Lane 1:
45S isolated from RB301 by depletion of RbgA; lane 2: mature 50S subunit isolated from
RB418 by depletion of IF2; lane 3: 44S complex isolated from RB1055 (rplF-R3C) and lane 4:
44S complex isolated from RB1068 (rplF-T68R). L6 protein was confirmed by LC/MS/MS. All
complexes were analyzed on 16% Tricine gel. C. 44S complex isolated from RB1068 contains
ribosomal protein L16. Lane 1: 45S isolated from RB301 by depletion of RbgA; lane 2: mature
50S subunit; lane 3: 45S complex isolated from RB1043 (rbgA-F6A); lane 4: 44S complex
isolated from RB1055 (rplF-R3C) and lane 5: 44S complex isolated from RB1068 (rplF-T68R).
L16 protein was confirmed by LC/MS/MS. All complexes were analyzed on 12% Bis-Tris gel.

140

Figure 4.7 Measurement of GTPase activity of RbgA in the presence of 44S intermediate from
suppressor strains.
The intrinsic GTPase activity of RbgA (lane 1) was determined by incubation of 2µM RbgA
protein with 200µM GTP for 15 minutes at 37°C. Stimulation of GTPase activity was measured
by incubation of 100nM RbgA protein with 100nM of mature 50S subunit (lane 2); 45S complex
isolated from RbgA depleted cells (lane 3); 44S intermediate isolated from suppressor strain
RB1051 (lane4); 44S intermediate isolated from suppressor strain RB1055 (lane5); 44S
intermediate isolated from suppressor strain RB1057 (lane6); 44S intermediate isolated from
suppressor strain RB1063 (lane7); 44S intermediate isolated from suppressor strain RB1065
(lane8) and 44S intermediate isolated from suppressor strain RB1068 (lane9). The values
represent the average of three independent experiments and the error bars represent the S.D.

141

Figure 4.8 Mutations in L6 protein affect subunit joining/interaction.
Ribosome profiles of strains expressing mutated L6 protein [RB1123 (rplF-R70P) is depicted
here as an example] show a higher concentration of individual ribosomal subunits and lower
concentration of 70S ribosomes compared with ribosome profile of wild type cells. The X-axis
indicates the direction of the profiles from the bottom of the gradient (25%) to the top of the
gradient (10%). The Y-axis depicts absorbance at 260nm, which is equivalent for both plots
depicted. Dashed lines indicate the migration of the 70S, 50S and the 30S complexes in the
gradient.

142

Figure 4.9 Mutations in L6 protein affect subunit joining/interaction.
Ribosome profiles of strains expressing mutated L6 protein RB1125 (panel 2), RB1131 (panel 3)
and RB1133 (panel 4) show a higher concentration of individual ribosomal subunits and lower
concentration of 70S ribosomes compared with ribosome profile of wild type cells (panel 1). The
X-axis indicates the direction of the profiles from the bottom of the gradient (25%) to the top of
the gradient (10%). The Y-axis depicts absorbance at 260nm, which is equivalent for all plots
depicted. Dashed lines indicate the migration of the 70S, 50S and the 30S complexes in the
gradient.
143

Figure 4.10 Analysis of ribosomal proteins L6 and L16 in 50S subunits that accumulate in
strains expressing rplF substitutions in wild-type background.
A. Ribosomal protein L6 is stably integrated in 50S subunits isolated from strains expressing
mutation L6 protein. Lane 1: 50S isolated from RB1123 (rplF-R70P); lane 2: 50S subunit
isolated from RB1125 (rplF-R3C); lane 3: 44S complex isolated from RB1055 (rplF-R3C); lane
4: 50S subunit isolated from RB1131 (rplF-G5S) and lane 5: 50S subunit isolated from RB1133
(rplF-T68R). L6 protein was confirmed by LC/MS/MS. All complexes were analyzed on 16%
Tricine gel. B. 50S subunits also stably integrate ribosomal protein L16. Lane 1: 45S isolated
from RB301 by depletion of RbgA; lane 2: mature 50S subunit; lane 3: 45S complex isolated
from RB1043 (rbgA-F6A); lane 4: 50S subunit isolated from RB1123 (rplF-R70P); lane 5: 50S
subunit isolated from RB1125 (rplF-R3C); lane 6: 50S subunit isolated from RB1131 (rplFG5S) and lane 7: 50S subunit isolated from RB1133 (rplF-T68R). L16 protein was confirmed by
LC/MS/MS. All complexes were analyzed on 12% Bis-Tris gel.

144

Figure 4.11 In vitro maturation of large subunit intermediates.
(A) in vitro maturation of 44S intermediate from RB1055. Ribosome profile from cell lysate of
strain RB1055 expressing mutated L6 protein (R3C) and RbgA-F6A protein after incubation at
0°C (blue) and 37°C (red) for 60 minutes. The profiles show a maturation of the 44S subunit to
the 50S subunit and a ~100% increase in the levels of 70S ribosomes after incubation at 37°C.
(B) in vitro maturation of 45S intermediate from RB1043. Ribosome profiles from cell lysate of
strain RB1043 expressing RbgA-F6A protein and wild-type L6 protein after incubation at 0°C
(blue) or 37°C (red) for 60 minutes. The profiles show a moderate ~10% increase in the level of
70S ribosome after incubation at 37°C. The X-axis indicates the direction of the profiles from the
bottom of the gradient (43%) to the top of the gradient (18%). The Y-axis depicts absorbance at
260nm, which is equivalent for both plots depicted. Dashed lines indicate the migration of the
70S, 50S, 44S and the 30S complexes in the gradient.

145

Figure 4.12 Interactions between L6 protein with the 50S ribosomal subunit.
Crystal structure of 50S subunit from E. coli (PDB ID :2AW4) with the late binding ribosomal
proteins (as indicated in figure) missing from intermediate 45S particles are shown in left panel.
Surface representation of all proteins are shown while RNA is shown in ribbon form. L6 binding
region including helix 97(colored magenta) is shown in magnified view in right panel. The
residues which are mutated in suppressor strains are colored in red at the N terminal of L6
protein.

146

Figure 4.13 Proposed model for the role of RbgA in the mediating productive h97-L6
interactions during the assembly of large ribosomal subunit.
RbgA is an essential protein and play a role in establishing correct L6-helix 97 interactions. An
intermediate (depicted as A) first bind to RbgA (depicted as B) that make the correct
incorporation site for L6 binding. After RbgA binding L6 joins the complex (C) which prepare
the binding site for binding of other late binding ribosomal proteins. Once the complex is
complete (D) the mature subunit stimulate the GTP hydrolysis activity of RbgA, that convert
GTP bound form of RbgA to GDP bound form which do not have affinity for 50S, as a result
RbgA in GDP bound form leaves the complex. In absence of RbgA, L6 binds to the intermediate
and forms 45S particle (I), but this complex cannot proceed further without the presence of
RbgA probably due to unproductive interactions between RbgA and L6. RbgA binding reverses
this interactions and assembly proceeds further as explained. (RbgA binding site is not known
the figure shows hypothetical binding site just for imagining)

147

Figure 4.14 Conserved interactions between L6 and helix 97 of 23S rRNA (ScL9-h97)
A. Crystal structure of 60S subunit from Saccharomyces cerevisiae (PDB ID: 3U5D and 3U5E)
with the positions of ribosomal protein L9 (ScL9, homolog of bacterial ribosomal protein L6,
cyan) and ribosomal protein L10 (homolog of bacterial ribosomal protein L16, green)
highlighted. B. A magnified view of the interaction between ScL9 (cyan) and h97 (magenta) is
shown. Structure of ribosomal protein L6 from E. coli (EcL6, blue) from 50S subunit (PDB ID:
2AW4) is superimposed on ScL9 (cyan) and the residues mutated in B. subtilis suppressor strains
are shown in red.

148

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153

CHAPTER 5
Summary and significance

154

Introduction
While the mechanistic details of ribosome structure and function have been researched
extensively, relatively less is known about how ribosomes are assembled in vivo[1-4]. For most
of the past 40 years research in the field of ribosome assembly has been focused on assembling
functional ribosomal subunits from its free purified components in vitro[5-8]. This has been
tremendously beneficial in uncovering the core rRNA and r-proteins required for optimal
ribosome function and the development of assembly maps for both 50S and 30S subunits[9-13].
However, partly due to the success of reconstitution experiments in vitro, research on in vivo
ribosome assembly has lagged behind. Even though the early in vitro reconstitution experiments
were highly successful in their desired aim of obtaining full functional ribosomal particles from
the constituent rRNA and r-proteins in vitro, the likely involvement of assembly factors for in
vivo ribosome assembly was hypothesized. In spite of this early realization, only over the last
decade have several assembly factors been identified and only a few of them have been
characterized functionally[14-16]. While several proteins from different classes such as
helicases, chaperones, modification enzymes, have been implicated in assembly, GTPases
feature prominently in the list and are universally required for ribosome assembly with several
factors implicated in assembly of bacterial, chloroplast and mitochondrial ribosomes [17-21].
Our current knowledge of different GTPases involved in ribosome assembly in bacteria was
discussed in chapter 1. These studies have mostly focused on ribosome assembly defects, growth
phenotypes linked to the depletion or mutation of ribosome assembly GTPases and only recently
research studies have focused on the direct interaction between these GTPases and few in vivo
ribosome assembly intermediates have been identified and characterized. In addition, most of the
assembly associated GTPases have not been biochemically characterized and relatively little is
155

known about their GTPase activity, catalytic mechanism and its role in assembly. The following
sections describe the significant results from the thesis and discuss the role of RbgA, an essential
GTPase in B. subtilis and its role in assembly of the 50S subunit, with an emphasis on its GTPase
activity and interaction with ribosomal subunits and intermediate particles.

Role of GTPase activity
Ribosome biogenesis GTPaseA protein, RbgA is an essential GTPase required for the biogenesis
of the 50S subunit in Bacillus subtilis[22,23]. Depletion of RbgA results in the absence of mature
50S subunits and accumulation of a large complex that migrates at 45S that lacks key ribosomal
proteins [22,23]. Research presented in chapter 3 indicated that histidine9 is likely the catalytic
residue required for GTPase activation of RbgA in the presence of the 50S subunit. This residue
in RbgA, is part of a PGH motif contained within the N-terminus of RbgA contains a stretch of
amino acid residues that is highly conserved in RbgA homologs including eukaryotic and human
homologs. In addition, the catalytic mechanism of RbgA seems similar to the mechanism of
GTPase activation of EF-Tu upon interaction with the 50S subunit and raises an interesting
possibility that assembly-associated GTPases interact with the ribosomal subunits similar to
translation-associated GTPases.
Biochemical characterization of RbgA and its interaction with the 45S complex and the 50S
subunit (Chapter 2) demonstrated that GTPase activity of RbgA is stimulated ~60 fold in the
presence of the 50S subunit and initially supported a model in which RbgA utilizes its GTPase
activity to exit a mature 50S subunit. Interestingly, strains expressing RbgA mutants with
reduced GTPase activity (chapter 3) accumulated a 45S complex similar to RbgA-depleted cells
156

suggesting that GTPase activity may play a role in maturation of the 50S subunit and may not
simply be required to exit a mature subunit. It is possible that L16 (part of the mature 50S
subunit but not the 45S complexes) could be the trigger for GTP hydrolysis or a local rRNA
structure specific to the mature subunit could be required for hydrolysis.

Interaction with rRNA
We identified a RNA binding domain (the ANTAR domain) in the C-terminal domain of RbgA.
The ANTAR domain is utilized by transcription antiterminator proteins to bind to stem-loop
structures in mRNA for translational control. This provides an interesting possibility of direct
interaction between rRNA and RbgA. While footprinting assays have indicated that RbgA
protects specific bases in rRNA there is so far no evidence of a direct interaction between RbgA
and rRNA[23]. The structural complexity of 23S rRNA does not allow us to test specific stemloop structures for association with RbgA. However future experiments utilizing chemical
probing might narrow the regions/domains of 23S rRNA that could be tested for interaction with
RbgA. It is an interesting possibility to consider that ribosome assembly GTPase interacts
directly with rRNA and impacts its secondary and/or tertiary structure. The highly structured and
long rRNA molecule likely misfolds and falls into kinetic traps during assembly[24]. Evidence
from reconstitution experiments demonstrates that several steps require incubation at high
temperatures to form a functional ribosomal subunit. It has been suggested that these incubation
steps alter the structure of the rRNA and allow it to escape from the aforementioned kinetic
traps. A similar functional role may be performed by assembly factors during in vivo assembly.
While RNA helicases are the likely candidates for altering rRNA structure due to their RNA-

157

dependant-ATPase activity evidence of direct interaction between GTPases and rRNA structure
would raise the possibility that GTPases could also play a role in rRNA modeling perhaps with
the involvement of GTP hydrolysis.

RbgA and assembly of the 50S subunit
Though results from chapter 2 and chapter 3 provided insights into the structure and function of
RbgA one question that remains unanswered is how the GTPase activity in the N-terminal
domain of RbgA is linked to the C-terminal ANTAR domain in the overall function of RbgA in
ribosome assembly. To test the communication between these two domains an RbgA-dependent
50S maturation assay is required. Research on suppressors of RbgA-F6A presented in chapter 4
could potentially aid in the development of an assay that can probe RbgA dependent maturation
of the 50S subunit. Our results show that the suppressor strains accumulated a novel large
complex that migrates at ~44S and is distinct from the 45S complex. Further, preliminary
maturation assays demonstrate the 44S particles from RbgA-F6A suppressor strains can mature
into a 50S subunit. Future experiments to achieve RbgA-dependent maturation are underway.
The success of such an assay would help elucidate the role of RbgA in assembly of the 50S
subunits and for the first time demonstrate GTPase facilitated in vitro ribosome assembly.
Further, should such an assembly be possible at physiologically relevant temperatures, as
opposed to the elevated temperature currently required for in vitro reconstitution, this would
constitute a particularly compelling demonstration of the role of GTPases in ribosome assembly
in vivo. Such a factor dependent in vitro assembly has previously been shown for the chaperone
protein DnaK but conflicting reports exist about its role in ribosome assembly[25,26]. An assay

158

that tests RbgA function in 50S maturation could also be used to elucidate the interaction
between N-terminal domain and ANTAR domain of RbgA during ribosome assembly.
Finally, structural studies of the 44S complex would be another exciting future avenue of
research. In particular, the structural differences between the 44S, the 45S and the mature 50S
subunit would highlight the difference in local rRNA structure and r-protein positions and
provide insight into the events at a late stage of ribosome assembly.

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