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HE. J

MSU Is An Afflrmdlvo Action/Equal Opponunity Institution
cmm1

 

 

IDENTIFICATION OF GENES REGULATED BY dnaA PROTEIN
IN Escherichia c211

By

Qingping Wang

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1989

I

ABSTRACT

IDENTIFICATION OF GENES
REGULATED BY dnaA PROTEIN IN Escherichia $911

By

Qingping Wang

dnaA protein is an essential protein for initiation of DNA replication from
the Escherichia 9.011 chromosomal origin, mg. It binds to the 9119 region by
recognizing a nine base-pair sequence 'I'l‘AT(A/C)CA(A/C) A. The binding
induces localized strand melting which promotes subsequent events in the
initiation process.

The dnaA protein binding sequence is also present in promoter regions of
several genes. This project was undertaken to test the possibility of dnaA
protein as a regulatory protein of gene expression. It was observed that dnaA
protein binds to promoter regions of genes dnaA, mH, 291A, 1133, and, and a
gene located near oriQ encoding a 16 KDa protein whose function is unknown
(referred to as 16 KDa gene). It also binds to some unidentified chromosomal
fragments. The binding resulted in transcriptional repression of dnaA, 11911,
and the 16 KDa gene. The effect of binding at other sites was not
conclusively determined. A model was proposed to explain the regulatory
function of dnaA protein.

In addition, a new sigma factor was discovered while working with the
mall gene. It has a molecular weight of 24 KDa and promotes transcription
from one of the three mQH promoters.

To
My Parents and Husband

ACKNOWLEDGMENT

I would like to express my appreciation to Dr. Jon Kaguni for his
guidance and encouragement through the years. I also owe thanks to Dr.
Laurie Kaguni for her suggestions and advice. I wish to thank members of
my guidance committee, Drs. Susan Conrad, Jerry Dodgson, Arnold Revzin
and John Wang for their advice and invaluable time.

I would like to acknowledge the support of my coworkers, Dr. Deog Su
Hwang, Dr. Cathy Wernette, Ted Hupp, Kevin Carr, Shelly Houston, David
Siemieniak, Matthew Olson, David Lewis, Rhod Elder, Philis Yang, and Zhun
Lu. I am also grateful to my Chinese friends here who made the years more
enjoyable.

I wish to thank my parents for their support and encouragement along

the way. Finally, my appreciation goes to my husband for his help,
understanding and support.

TABLE OF CONTENTS

page
LIST OF TABLES ..................................... ix
LIST OF FIGURES ..................................... x
ABBREVIATIONS ..................................... xii
INTRODUCTION ...................................... 1
Chapter I LITERATURE REVIEW .................... 6
I. dnaA Protein and Replication of
the E. 99.11 Chromosome ......................... 7
A. Genetic and Physiological Studies of DNA
Replication and dnaA Protein ................ 8
1. DNA Replication and Cell Growth ........... 8
2. Regulation of Initiation and Function of
dnaA Protein ....................... 11
a. Proteins Required for Initiation
from 9:19 ..................... 11
b. dnaA Protein and the Frequency of
Initiation ..................... 14
c. Other Factors Involved in the
Control of DNA Replication ......... 16
B. Biochemical Studies on 9:19 Replication ............ 18
1. Cloning of the 911°C. Region ................ 18
2. DNA Replication Proteins ................. 21
3. dnaA Protein .......................... 26
4. Mechanism of Initiation from mg ........... 27
C. Other Functions of dnaA Protein ................. 32
1. Involvement in Replication of Plasmids
pSClOl, R1, R100, F, P1 and ColE1 ....... 32
2. Repressor Function in Gene Expression ........ 32
II. The Heat-shock Response in E. 51911 ................... 34
A. Heat-shock Proteins ......................... 34
B. The Heat-shock Regulatory Protein, Sigma32 ........ 36
C. Regulation of the Heat-shock Response ............ 37
III. Objectives of the Project .......................... 38

References ......................................

Chapter II TRAN SCRIPTIONAL REPRESSION OF THE dnaA
GENE OF Escherichia 5:911 BY dnaA PROTEIN . . . .

Abstract ........................................
Introduction .....................................

Materials and Methods ..............................
Plasmids and DNAs ...........................
Enzymes ...................................
DNA Binding ................................
Transcription Assays ...........................
Radioactive Labelling of DNA .....................

Results ........................................
dnaA Protein Specifically Inhibits Transcription
from the dnaA Promoters ...................
dnaA Protein Specifically Inhibits Transcription
from the 16 KDa Promoter ..................
Specific Binding of dnaA Protein to
Restriction Fragments .....................

Discussion ......................................

References ......................................

Chapter III TRAN SCRIPTIONAL REPRESSION OF THE mgfl
GENE OFEacheflghiamliBdeaAPROTEIN . . . .

Abstract ........................................
Introduction .....................................

Materials and Methods ..............................
Bacterial Strains and Plasmids ....................
Enzymes ...................................
Radioactive Labelling of DNA .....................
DNA Binding Assays ...........................
Run-off Transcription Assays .....................
RNA Preparation .............................
Sl Nuclease Assays ...........................

49
50
51
54
54
55
55

56
57

58

92

Results ....................................... 101

dnaA Protein Binds Specifically to Restriction fiagments

 

Containing the mnH Promoter Region .......... 101
dnaA Protein Binds to the dnaA Boxes in the 132911
Promoter .............................. 106
dnaA Protein Inhibits Transcription From Two of the
Three :1ng Promoters in vitro ................ 111
dnaA Protein Inhibits mH Transcription in vivo ....... 118
Discussion ...................................... 126
References ...................................... 130
Chapter IV A NOVEL SIGMA FACTOR INVOLVED IN
EXPRESSION OF THE mQH GENE OF
M mli ......................... 134
Abstract ........................................ 135
Introduction ..................................... 136
Materials and Methods .............................. 138
Bacterial Strains and Plasmids .................... 138
Enzymes ................................... 138
Elution of Proteins from SDS-polyacrylamide Gels ....... 139
Run-off Transcription Assays ..................... 140
Induction of the Stringent Response ................ 140
Detection of in vivo Synthesized Transcripts ........... 141
Results ........................................ 142
Identification of a Factor Required for mHBP
Transcription ........................... 142
The mH3P Transcription Factor Is a Heat-stable
Sigma Factor ........................... 147
The mH3P Transcription Factor Is a Protein of 24 KDa . . 147
The 24 KDa Protein Acts as a Sigma Factor ........... 152
Sigma 24 Is Not Identical to Stringent Starvation Protein . 155
Discussion ...................................... 159
References ...................................... 162
Chapter V PREFERENTIAL BINDING OF dnaA PROTEIN T0

DNA FRAGMENTS CONTAINING PROMOTER
REGIONS OF 9.0.15, HEB, AND and, AND

FRAGMENTS CONTAINING SOME

UNIDENTIFIED SITES .................... 165

vii

Abstract ........................................ 166

Introduction ..................................... 167

Materials and methods .............................. 169

Strains, DNAs, and Enzymes ..................... 169

Nitrocellulose Filter Binding Assays ................ 169

Results ........................................ 171
dnaA Protein Recognition Sequences in Promoter Regions

of 991A. mgr-B, and nrd ..................... 171

Binding of dnaA Protein to the Three Promoter Regions . . . 171
Chromosomal DNA Fragments Selectively Bound

by dnaA Protein ......................... 181

Discussion ...................................... 189
References ...................................... 199
Chapter VI SUMMARY AND PERSPECTIVES ................. 202

LIST OF TABLES

page
Chapter V
1. Number of sites of the dnaA binding sequences
in the sequenced E. 9211 genome. ................... 190
2. Locations of TTATCCACA Sites. ...................... 192
3. Locations of 'I'I‘ATACAAA Sites ...................... 193
4. Locations of 'l'I‘ATC CAAA Sites ...................... 194
5. Locations of TI‘ATACACA sites ...................... 195

LIST OF FIGURES

page

Chapter I

1.
2.
3.

Schematic presentation of the cell division and

chromosome replication cycle.. ....................
Consensus sequence of the minimal origin of

the bacterial chromosome. .......................
Specific priming systems for M13, G4, ¢X174 DNAs

coated with SSB. .............................

4. A model for initiation of replication from 9:19. ............

Chapter II

1.
2
3.

4
5.
6
7

Map of the dnaA and mmI-I promoter regions and
coding regions contained in the 945 bp
EcoRI fragment ..............................

. Map of the gene encoding the 16 103a protein and

adjacent 2.an gene ............................
Run-off transcription using the 945 bp EcoRI

restriction fragment. ..........................
Run-ofl' transcription using the 569 bp HaeIII-EcoRI

restriction fragment. ..........................
Run-off transcription from the B-lactamase promoter

and from the lacUV5 promoter .....................
Run-ofl‘ transcription using the 328 bp BamHI-Hinfl

restriction fragment. ..........................
In vitro transcription using the 414 bp HpaII

restriction fragment containing the promoter region

for the 16 KDa protein. .........................
Preferential binding of dnaA protein to a restriction

containing the dnaA promoters. ...................
Preferential binding of dnaA protein to a restriction

fragment containing the 16 KDa promoter. ............

Chapter III

1.
2.

Physical map and DNA sequence of the 132911 promoter .....
Preferential binding of dnaA protein to a restriction

fragment containing the moi-I promoter region. .........

61

69

75

81

102
104

3. Preferential binding of dnaA protein to restriction
fragments containing the dnaA promoter region,

or the 11911 promoter region. ..................... 107
4. dnaA protein binds to both dnaA boxes in

the mold promoter region. ....................... 109
5. Run-off transcription assays with DNA fragments

containing the 132911 promoters. .................... 113
6. dnaA protein inhibits transcription

from mHBP and moH4P. ....................... 115
7. Inhibition of mfl4P transcription by overproduction

of dnaA protein in vivo. ......................... 119
8. Inhibition of mHSP and [pol-MP transcription

by dnaA protein after a temperature shift. ............ 123

Chapter IV

1. Physical map of the mall promoter region ............... 143
2. Run-off transcription assays with preparations

of RNA polymerase. ........................... 145
3. The sigma factor which confers moHSP recognition

is heat stable. ............................... 148
4. Isolation of a novel sigma factor by electroelution

from an SDS-polyacrylamide gel. ................... 150
5. Competition between the 24 KDa protein and 07°

for core enzyme. .............................. 153
6. Repression of mHSP under amino acid starvation. ......... 157

Chapter V

1. Map of the promoter regions of mm,

m, and nrd. ............................... 172
2. Preferential binding of dnaA protein to a restriction

fragment containing the MA regulatory region. ........ 174
3. Binding of dnaA protein to restriction fragment

containing the m3 promoter region. ................ 17 7
4. Preferential binding of dnaA protein

to the nxrB promoter region. ..................... 179
5. Binding affinity of dnaA protein to DNA fragment

containing the nnd promoter region. ................ 182
6. Preferential binding of dnaA protein to

chromosomal DNA fragments. ..................... 184
7. Binding afiinity of a 700 bp cloned fragment. ............. 187

dNTP

EDTA
HEPES

KDa

mg plasmid
PIPES

SDS

SSB

E6”

Ea”

ABBREVIATIONS

base pair

deoxyribonucleotide

dithiothreitol

ethylenediamine tetraacetic acid
4-(2-hydroxethy1)—1-piperizineethane sulfonic acid
kilo-base pair

kilo-dalton

plasmid containing the gn'Q sequence
piperazine-N,N’-bis(2-ethanesulfonic acid)
sodium dodecyl sulfate

single-stranded DNA binding protein
RNA polymerase with sigma70

RNA polymerase with sigma32

INTRODUCTION

Escherichia £911 is probably the best studied organism. DNA replication of
its chromosome has been a major subject of study for over thirty years. E. c911
has a single circular chromosome of about 4,000 kilo-base pairs (kb) (1-3).
Replication of the chromosome starts at a specific site, the chromosomal origin
or mic, proceeds bidirectionally with equal velocity, and terminates at the
opposite position on the circle (4-6).

dnaA, the first genetic locus identified whose function is involved in
initiation of DNA replication (7), encodes a protein that specifically binds to
the replication origin (8). The binding promotes subsequent events in the
initiation process to occur (9). Physiological studies indicate that initiation of
DNA replication is tightly coupled to cell growth. It occurs at a critical
DNA/cell mass ratio. dnaA protein plays an key role in determining the
initiation mass.

dnaA protein also binds to promoter regions of its own gene and the gene
encoding a 16 KDa protein of unknown fimction which is located adjacent to
ma (8). In vivo data indicate that dnaA protein represses transcription from
the dnaA promoters and from the 16 KDa promoter (10-12). DNA fragments
bound by dnaA protein share a nine base-pair consensus sequence
'I'I‘AT(A/C)CA(A/C)A (8). This sequence is also found in promoter regions of
other genes including moH, palA, m3 and nzd (13-16). Based on an

assumption that dnaA protein recognizes the above consensus sequence and

2
binds to it, and that binding of a protein at the promoter region of a gene
will influence its expression, this project was undertaken to investigate the
possibility of dnaA protein regulating expression of other genes. This
regulation by dnaA protein may play an important role in coordination of
DNA replication to cell growth.

At the time the project was started, in vivo data suggested that dnaA
protein represses expression of its own gene and of the 16 KDa gene (10- 12).
However, these in vivo experiments do not address whether dnaA protein
alone is sufiicient for the repression. In addition, an inherent problem of in
vivo experiments is that one cannot distinguish a primary effect of dnaA
protein from a secondary effect. Since these are very critical questions to the
project, in vitro experiments were performed to establish that dnaA protein
repressed expression of the dnaA and the 16 KDa genes with purified
enzymes. Transcripts from the dnaA promoters and from the promoter of the
16 KDa gene were observed in run-ofi‘ transcription assays as expected.
Addition of purified dnaA protein resulted in specific inhibition of these
promoters. The degree of repression was well correlated with the extent of
binding of dnaA protein to the promoter regions in filter binding assays.
These results demonstrated that binding of dnaA protein directly inhibits
transcription fi'om the dnaA promoters and from the 16 KDa promoter. This
work was published in MOLECULAR and GENERAL GENETICS (1987) 202.
518-525.

Two copies of the dnaA protein recognition sequence are present in the

promoter region of the mall gene (13). The mH gene encodes a sigma factor

3
of RNA polymerase which recognizes promoters of heat shock genes. Heat ‘
shock proteins of E. 9911 are involved in many macromolecular processes
including DNA replication, RNA synthesis, and protein processing. The
mechanism of action remains to be revealed. Using filter binding assays and
DNaseI protection assays, dnaA protein was observed to bind to the two nine
base-pair consensus sequences in the 131911 promoter region. The binding
resulted in transcriptional repression from two of the three promoters of the
@311 gene both in vivo and in vitro. Regulation of the 119171 gene by dnaA
protein suggests that synthesis of heat shock proteins which are required for
normal cell growth is coordinated with initiation of DNA replication. This
work was published in THE JOURNAL OF BIOLOGICAL CHEMISTRY
(1989) 264, 7338-7344.

In examining transcription of the anti promoters, it was observed that
several sigma70 RNA polymerase preparations were able to transcribe from
two of the three @211 promoters. Interestingly, one preparation resulted
additionally in transcription from the third promoter mnH3P. Reports from
others indicate that this promoter is not recognized either by sigma70 or
' sigma32 (17). Transcription from this promoter must require a positive
regulatory protein or a new form RNA polymerase. In pursuing this matter, a
new sigma factor was discovered which is required for transcription from
mH3P. This work is in press in the JOURNAL OF BACTERIOLOGY (1989).

The presence of dnaA protein recognition sequences in promoter regions
of DQJA, m3, and nzd resulted in binding of dnaA protein to these sites.
mlA, m3, and nnd encode DNA polymerase I, uvrB protein, and

4

ribonucleotide reductase, respectively. The former two are involved in DNA
repair and the later is required for the conversion of ribonucleotides to
deoxyribonucleotides. The efi‘ect of binding on transcription from promoters of
these genes was inconclusive.

In addition to these sites, dnaA protein was also observed to
preferentially bind to unidentified chromosomal fragments from E. 99.11.

This thesis is composed of five chapters. The first chapter is a review of
the literature on studies of DNA replication and heat shock response in E.
mli. The following chapters are derived from publications in MOLECULAR
and GENERAL GENETICS (1987) 209, 518-525 (chapter II); THE JOURNAL
OF BIOLOGICAL CHEMISTRY (1989) 26.4, 7338-7344 (chapter III); and the
JOURNAL OF BACTERIOLOGY (in press) (chapter IV). Chapter V
summarizes the unpublished studies on the binding affinity of dnaA protein to
promoter regions of mm, nyrB, and, and an unidentified chromosomal
fragment.

10.
11.

13.

14.

15.

16.

17.

REFERENCES

Jacob, F., and Wollman, E. L. (1961) Sexuality and the genetics of
bacteria. Academic Press, New York.

Cairns, J. (1963) J. M01. Biol. 6, 208-213.
Lewin, B. (1983) Gene. John Wiley & Sons.

Prucott, D. M., and Kuempel, P. L. (1972) Proc. Nat. Acad. Sci. USA
69, 2842-2845.

Louarn, J., Funderburgh, M., and Bird, R. (1974) J. Bacteriol. 120, 1-
5.

Marsh, R. C., and Worcel, A. (1977) Proc. Nalt. Acad. Sci. USA 74,
2720-2724.

Hirota, Y., Mordoh, J., and Jacob, F. (1970) J. Mol. Biol. 53, 369-387.
Fuller, R. S., Funnell, B. E., and Kornberg, A. (1984) Cell 38. 889-
900.

Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755.
Braun, R. E., O’Day, K., and Wright, A. (1985) Cell 40, 159-169.

Atlung, T., Clausen, E. S., and Hansen, F. G. (1985) Mol. Gen. Genet.
200, 442-450.

Lother, H., Kolling, R., Kucherer, C. and Schauzu, M. (1985) EMBO J.
4, 555-560.

Landick, R., Vaughn, V., Lau, E. T., VanBogelen, R. A., Erickson,. J.
W., and Neidhardt, F. C. (1984) Cell 38, 175-182.

Joyce, C. M., Kelley, W. S., and Grindley, N. D. F. (1982) J. Biol.
Chem. 257, 1958-1964.

Sancar, G. B., Sancar, A., Little, J. W., and Rupp, W. D. (1982) Cell
28, 523-530.

Carlson, J., Fuchs, J. A., and Messing, J. (1984) Proc. Natl. Acad. Sci.
USA 81, 4294-4297.

Erickson, J. W., Vaughn, V., Walter, W. A., Neidhardt, F. C., and
Cross, C. A. (1987) Genes & Develop. 1, 419-432.

Chapter I
LITERATURE REVIEW

LITERATURE REVIEW

1. dnaA Protein and Replication of the E. 99.11 Chromosome

Ever since DNA was proven to carry genetic information four decades ago
(1), replication of DNA has received a lot of attention. In 1953, Watson and
Crick proposed the double stranded DNA model based on X-ray diffraction
data (2). The model predicted that DNA is replicated in a semiconservative
fashion, which was confirmed experimentally by Meselson and Stahl in 1958
(3). Since these discoveries, a considerable amount of research efi‘ort has been
focused on DNA replication in E. 9911. Early genetic studies indicate that the
E. 9911 chromosome is a closed circular duplex DNA (4). This is supported by
autoradiographic studies performed by Cairns in 1963, who discovered that
the chromosome exists as a closed circle of about 1000-1400 pm in length by
labeling the chromosome with tritiated thymidine (5). This length was
determined later to be about 4000 kb (6). Cairns also observed only one
replication bubble on each chromosome suggesting that the replication is
initiated at only one point (5). Later studies indicate that DNA replication
starts at a fixed position on the chromosome, mg, proceeds bidirectionally
with equal velocity, and terminates at a point, m opposite from mg on the
circular chromosome (6-8).

Jacob and coworkers, based on limited and circumstantial evidence of

DNA replication of E. 99.11 chromosome, F factor, and of phages, proposed a

8
replicon model in 1963 to explain regulation of DNA replication (9). The
model proposed the following. 1. Any independent replicating DNA consists of
a replicon, which contains a operator of replication, or replicator. The
replicator is a specific element on the chromosome which allows an initiator
protein to recognize and to promote DNA replication. 2. An initiator protein is
required for initiation of DNA replication from a replicator of a specific
replicon. The synthesis of the initiator is controlled so that it is accumulated
to allow the replication to occur in a precisely timed manner. This model,
formulated similarly to the repressor-operator model in the regulation of
transcription, is demonstrated to be largely correct afier 25 years of study.

A. Genetic and Physiological Studies of DNA Replication and dnaA Protein

1. DNA Replication and Cell Growth

The doubling time of E. c911 varies from 20 minutes to 150 minutes
depending on nutrient conditions (10). The size of fast growing cells are larger
than those growing slower. Small cells usually contain one to two genome
equivalents of DNA, while large, fast growing cells can have more than four
genome equivalents of DNA. DNA content per cell changes as the cell size
varies resulting in a relatively unbiased ratio of DNA to cell mass
independent of growth rate (10).

Regulation of DNA replication in relation to the cell division cycle was
studied in E. £9.11 by Helmstetter and C00per (11, 12) (Figure 1). DNA

synthesis was measured by pulse labeling an undisturbed population of

Figure 1. Schematic presentation of the cell division and chromosome
replication cycle (170).

The doubling time is assumed to be 90 minutes (a), 60 minutes (b), and
35 minutes (0). tn, doubling time; C, DNA synthesis period from initiation (ini)
to termination (ter) of chromosomal replication; D, time between termination

and cell division (div).

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1 1
exponentially growing cells. The amount of label was later correlated to cells
at difi‘erent stages in the division cycle by a filter elution technique. The rate
of DNA synthesis in cells at different stages was deduced from the amount of
label incorporated. Assuming that the rate of DNA synthesis is constant at
each replication fork, an increase in the rate was explained as an increase in
the number of replication forks, that is initiation of a new round of
replication. A decrease in the rate was interpreted as a termination of
existing replication forks. By measuring initiation and termination in closely
timed cell cycles, the following was concluded. 1. The time for a complete
round of replication is constant and requires about 41 minutes. The time
between the termination of replication and cell division is also a constant of
about 20 minutes. 2. New rounds of replication start at a time independent of
the cell age, but at a constant time before a given division occurs. 3. In
rapidly growing cells, new rounds of replication begin before the previous
round has completed. Individual chromosome in such cells contains multiple
forks. Further refined studies indicate that initiation begins at a fixed ratio of

cell mass to replication origin (13) (Figure 1).

2. Regulation of Initiation and Motion of dnaA Protein

a. Proteins Required for Initiation from 2:19,
Since DNA replication is regulated by the frequency of initiation, proteins
involved in initiation became the focus of study. Jacob’s initiator protein was

the apparent target to start with (9). A genetic approach was used to identify

12

genes whose products function in initiation of DNA replication. Since initiator
proteins are required for viability, mutations in initiator genes were selected
in temperature sensitive conditional mutants. These mutants were able to
synthesize DNA at permissive temperature. But at nonpermissive
temperature, such mutants could only finish the on-going replication forks
and were unable to initiate new rounds of replication. The first of such
mutants, (23146. was isolated and characterized by Hirota et al (14). This
locus was named dnaA, and was located at 83 minutes on the 100 minute
genetic map of the chromosome. The mutation was named dnaA46. Soon after
the discovery of dnaA46, other initiation mutants, with similar phenotypes
but located at difi'erent positions on the chromosome, were isolated. These loci
were named dnaB, dnaQ, dnaI, and M (15-18). Mutants of flush and M
were also found with a phenotype characteristic of genes involved in
elongation rather than in initiation (15, 104, 169). Mutations in final and
m were not extensively studied. Physiological studies indicated that protein
and RNA synthesis are also required for initiation (19-22). The products of
MMmddnaflwiflbemfenedtoasdnaAdnaB,anddnaC proteins,
respectively.

dnaA protein functions in an early stage of initiation. The finding by
Zyskind et al in 1977 that rifampin had a different efl'ect on dnaA and M
mutants suggested an order of action between these two gene products (23).
Mutants of dnaA or dnaQ held at nonpermissive temperature (42°C) for 1.5
generation time can express the initiation potential accumulated at 42°C when
shifted back to permissive temperature (30C). Rifampin blocks RNA synthesis

13
by inhibiting RNA polymerase. Addition of rifampin 10 minutes before
returning the culture back to 30°C completely abolished the potential to
initiate DNA synthesis in a dnaA mutant. N 0 effect was observed in a dnaC,
mutant. These results indicated that the interaction between dnaA protein
and RNA polymerase occurs before the function of dnaC.

Kung and Glaser arrived at the same conclusion with a different
approach (24). They constructed a double mutant carrying a heat-sensitive
dnaA allele and a cold-sensitive dnaC mutation. DNA synthesis proceeds
normally at 32°C but initiation is inhibited both at 20°C and 42°C. When a
culture growing at 32°C was first shifted to 20°C for some time before shifting
to 42°C, DNA synthesis stopped at 20°C slowly due to the defect in dnaC
protein; it resumed at 42°C for one round of replication even in the presence
of defective dnaA protein. This suggested that an initiation intermediate
formed by dnaA protein at 20°C can be extended by dnaC protein at 42°C.
When the culture was first shifted to 42°C and then to 20°C, DNA synthesis
did not resume at 20°C. These experiments indicated that the functions of
dnaA and dnaC protein are independent and that dnaA protein acts before
dnaC protein.

Extragenic suppressor studies confirmed the involvement of dnaA protein
in initiation of replication. According to the replicon model, an initiator
protein is required for a specific replicator (replication origin). If dnaA protein
is the initiator for 91:19, substituting mg with other replication origins should
suppress the replication defect in dnaA temperature sensitive mutants. Soon
after dnaA46 was isolated, Nishimura and Caro showed that integration of

14
the E. 94111 F factor into the chromosome suppressed the temperature
sensitivity of a dnaA46 strain (25). Similar results were obtained by Lindahl
et al. that a strain of M46 under control of prophage P2 became
temperature resistant (26). However, the integration sites of F factor or phage
P2 were shown to be crucial (27).

Another suppressor of dnaA dependent initiation was found in mh, the
gene encoding RN aseH which degrades RNA in a DNA-RNA hybrid. Initiation
of DNA replication from mic dependent on the dnaA gene product requires
protein synthesis (19-21). Mutant of mh gene can sustain continued DNA
replication in the absence of protein synthesis suggesting that an alternate
pathway of replication was utilized (28, 29). In 1983, Kogoma and von
Meyenburg showed that a mh mutation suppressed both mic deletion and
dnaA mutations (30). Their results were confirmed by studies from other
laboratories (31-34).

b. dnaA Protein and the Frequency of Initiation

Initiation potential accumulates in dnaA mutants shifted to
nonpermissive temperature (42°C). In an experiment performed by Tippe-
Schinder in 1978, a W strain was grown at 30°C and shified to 42°C for
various times (35). Initiation potential was assayed by DNA synthesis at 30°C
in the presence of chloramphenicol, which blocks synthesis of dnaA protein.
DNA synthesis at 30°C was observed to be directly related to the time that
the culture was held at 42°C. Further studies showed that this potential is

dependent on protein synthesis at 42°C and can only be expressed in

15
reversible mutants (36). These results indicate that dnaA protein synthesized
at 42°C, though not active at 42°C, can support initiation when returned to
30°C. Consistent with this conclusion is the observation that more than two
round of initiation occurred in cells shifted to 30°C after being held at 42°C
for 1.5 generations (37 -40).

In contrast to heat sensitive mutants, a cold sensitive dnaA mutant
overinitiates DNA synthesis at nonpermissive temperature (30°C) (41). This
mutant grows normally at 42°C but cannot form colonies at 30°C.
Measurements of DNA and protein synthesis indicated that mutant cells had
a normal DNA content at 42°C. Upon shifting to 30°C, the ratio of DNA to
protein increased up to 8 fold compared to the value at 42°C as a result of
increased DNA synthesis. The ratio did not change significantly in a wild type
control strain. Apparently, the temperature sensitivity of this mutant was due
to overinitiation. Similar lethal efi‘ects were also observed in other strains
that overinitiate DNA replication (42).

If dnaA protein controls the initiation frequency, overproduction of dnaA
protein from a plasmid carrying the dnaA gene should result in increased
initiation. Initial observations on the ratio of DNA to cell mass indicated that
the ratio was not significantly influenced by overproduction of dnaA protein
(43-45). This apparent dilemma was resolved when Atlung et a1. observed that
genetic markers near m9 were present at a higher frequency than the rest of
the genome as determined by hybridization to different marker DNA probes
(46). These results suggest that replication forks produced by overinitiation
were halted soon after leaving mg resulting in no substantial change in DNA

16
content. The presence of excessive replication forks at elevated levels of dnaA
protein was confirmed by measurement of DNA accumulation in the presence
of rifampin, which blocks initiation but allows existing replication forks to
proceed. A two- to three-fold increase in DNA accumulation was observed in a
dnaA protein overproducing strain (47). These results indicate that dnaA
protein is an important factor in regulation of initiation of DNA replication
from QIIQ-

c. Other Factors Involved in the Control of DNA Replication

Factors other than dnaA protein seem to be involved in the control of
DNA replication. Multiple initiations occurred after a reversible heat sensitive
dnaA mutant held at 42°C and returned to 30"C were separated by about 30
minutes (37-40). This separation time was independent of the mutations used
and is likely determined by some other limiting factors. One such factor could
be the state of methylation of the ma sequence. The 9:19, region contains an
extraordinarily high number of the sequence, GATC, which is recognized and
methylated by deoxyadenosine methylase (48, 49). E. can DNA is normally
fully methylated. It is only hemimethylated for a short time following
replication. The state of methylation is widely known to influence biological
properties of DNA (50). Studies of the 911:: sequence indicate that
hemimethylated DNA cannot support initiation (51-53). Methylation of mg
takes about 10 minutes after initiation (54). During this period, the ma
region remains attached to cell membrane and is later released (54).

Subsequent events might need to take place before another initiation can be

17
engaged in.

Another line of evidence indicating other factors are involved in the
control of initiation is based on studies of dnaA mutants at intermediate
temperatures (25°C-35°C) (55). Hansen et a1. noticed that the replication
activity of a dnaA46 strain was significantly reduced at temperatures above
35°C presumably due to inactivation of dnaA46 protein. At temperatures
between 25°C and 35°C, where inactivation of dnaA46 was not apparent, the
total initiation events per cell still decreased with increasing temperature.
Simultaneously, an increasing initiation potential was accumulated linearly
with temperature. This potential can be expressed at the same growth
temperature in the absence of protein synthesis. These results suggest that an
inhibitor of initiation, which is rapidly degraded in the absence of protein
synthesis, is produced at increasing levels between 25°C and 35°C. Similar
results were also observed by others (56).

Consistent with this finding is the following observation. In cultures of
dnaA46 and W grown at intermediate temperatures, addition of
chloramphenicol increased the rate DNA of synthesis (57, 35, 39). The amount
of increase was dependent on the growth temperature. In contrast to the
mutant cells, no increase in the rate of DNA synthesis was observed in the
wild type control indicating that the inhibitor was not normally present at a
significant level. Since dnaA protein is temperature sensitive in these strains,
and dnaA protein is currently known to regulate gene expression, it is highly
possible that dnaA protein represses the expression of this inhibitor, which

normally under tight control is overexpressed in dnaA mutants even at

18
intermediate temperatures. Unfortunately, this subject was not further
studied.

B. Biochemical Studies on ma Replication

Biochemical studies were made possible by two lines of research: the
cloning of the ma sequence; and the identification of replication proteins.
Using plasmid DNA carrying the m9 sequence, in vitro mic, replication
reactions were reconstituted with purified enzymes. The function of individual
proteins was then studied in detail.

1. Cloning of the ma Region

Genetic studies located ms: ‘at about 84 minutes on the 100 minute E.
99.11 chromosome between genetic markers asnA and 12ng (58). These latter
genes encode proteins involved in arginine synthesis and glucose metabolism,
respectively (58). The origin region was first isolated in a 9 kb EcoRI
fragment (59, 60). Subcloning of this fragment localized the origin to a 422
base pairs (bp) region and this region was sequenced (61, 62). By deletion
analysis of the 422 bp region, the origin function was located within a
minimum of 245 bp region (48).

Comparison of mg sequences in six related bacteria reveals several
significant features (Figure 2) (49). 1. The sequence GATC, the site for
deoxyadenosine methylase, is highly concentrated in the ma region. Only two
sites are expected in the 245 bp minimum region if GATC is present at

19

Figure 2. Consensus sequence of the minimal origin of the bacterial
chromosome (49).

The consensus sequence is derived from six bacterial origin sequence,
those ofE. mli.S-tnhimmium.Entemhamaem£enea.K.nnenmnniae.
Emma Mom, and Y. hamyi. The alignment of the six sequences is
such that the least number of changes are introduced into the consensus
sequence. In the consensus sequence, a large capital letter means that the
same nucleotide is found in all six origins; a small capital letter means the
nucleotide is present in five of the six sequences; a lowercase letter is used
when that nucleotide is present in three or four of the six bacterial origins
but only two different nucleotides are found at that site; and where three or
four of the four possible nucleotides, or two difi'erent nucleotides plus a
deletion, are found at a site, the letter n is used. Bold large capital letters
locate at positions 149, 242, and 267, where single-base substitutions produce
an griQ‘phenotype in E. £011- GATC sites are underlined in the consensus
sequence and certain E. 9911 restriction sites are noted. The minimal origin of
E. £911 is enclosed within the box. The numbering of the nucleotide positions
is that used for E. £911. and the upper left end is the 5’ end. The four dnaA
binding sequence are indicated as R1, R2, R3, and R4. The 13-mer repeats

are also indicated on the upper left corner.

20

 

 

 

 

 

 

 

 

 

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21
random. Eleven methylation sites are present in the E. ggli origin and eight
of them are conserved. The significance of these methylation sites may relate
to its involvement in initiation of DNA replication as discussed above. 2. Four
copies of a nine base pair sequence 'I'I‘AT(A/C)CA(A/C)A are also conserved.
This sequence is recognized and bound by dnaA protein. 3. The left side of
the minimum sequence contains three 13 base pair repeats which are also
highly conserved. The 13 base pair sequences are involved in early steps in
the initiation process. This and the function of the dnaA protein binding
sequence will be discussed later.

Initiation of mic plasmids is under a control similar to that of initiation
of the chromosome. The products of dnaA, dnaC, and other genes required for
the chromosomal replication are also needed for the plasmid replication (63).
However, the kinetics of mg plasmid replication seems to be different from
that of the chromosome. No coupling to initiation of chromosomal replication
was observed (64). Furthermore, unidirectional replication of some 9119
plasmids was observed, in contrast to the bidirectional replication of the
chromosome (65-67). The difi'erence between replication of the chromosome
and ma plasmids suggests that sequences outside the minimum region are
required for accessory functions.

2. DNA Replication Proteins

Three DNA polymerases, pol I, pol II, and pol III, exist in E. £9.11 (68).
The first efi‘ort in searching for enzymes involved in DNA replication resulted
in the discovery of pol I in the late fifties (69). Because of the assay system

22
used in pol I purification, the activity of pol II and pol III was not detected.
The fact that DNA replication in vivo was hardly affected by some mutations
in pglA, the gene coding for pol 1, indicated that other DNA replication
enzymes were present in E, 2911. By altering assay conditions, DNA
replication activity was found in ngA mutant extracts. This replication
activity was resolved into two distinct peaks in subsequent chromatography.
The two peaks were designated pol II and pol III. Pol III has a very high
processivity on primed single-stranded DNA template covered with single-
stranded DNA binding protein (SSB). pol III is responsible for chromosomal
replication. The function of pol I has been considered as gap filling because of
its low processivity. Much less is known about pol II. This enzyme seems to
be involved in DNA repair. All DNA polymerases require a primer
complementary to the template to provide a 3’-OH terminus for subsequent
elongation.

Other than DNA polymerases, many accessary proteins are involved in
DNA replication, mostly in primer synthesis. The major body of knowledge
about these proteins came from studies of replication of single-stranded DNA
phages M13, G4, and ¢X174 (Figure 3) (68). Genomes of these phages are
circular single-stranded DNAs of several kilo-bases (68). The priming and
replication process were expected to be relatively simple, since no topological
constrains are involved.

Replication of M13 is initiated by synthesis of a RNA primer by RNA
polymerase at a specific hairpin structure on the circular DNA (70, 71).
Polymerase III functions to extend the primer to full length. Pol I will remove

23

Figure 3. Specific priming systems for M13, G4, tXl74 DNAs coated with
SSB (68).

24

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POLYMERASE PRIMER
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25

the RNA primer and fill the small gap left by pol III. The ends of the newly
synthesized strand are then joint by DNA ligase. G4 replication uses a
different enzyme, primase, for primer synthesis (72-74). Primase can use both
ribonucleotides and deoxynucleotides as substrate (75-77). In a similar
manner, primase also recognizes a specific hairpin structure. The DNA
synthesis is the same as that of M13.

Replication of ¢XI74 is much more complicated than that of M13 and G4.
No single enzyme is able to prime DNA synthesis. A multi-protein complex,
the primosome, containing at least seven proteins, is required for primer
synthesis (78-80). The assembly of the primosome is initiated by binding of a
sequence-specific DNA binding protein, 11’, to the assembly site (81, 82). Other
primosomal proteins, n, n”, i, dnaC, dnaB, and primase, are then successively
assembled on n’ (80). When priming is uncoupled from DNA synthesis, 5-10
primers are synthesized on the circle (83), yet the primosome is still tightly
associated with the template (84), indicating the primosome has moved.
Detailed studies revealed that the primosome moves in the direction opposite
of primer synthesis (82). It was proposed that the primosome functions in
primer formation on the lagging strand during replication of a double
stranded DNA-the primosome moves along with the replication fork, while
primer formation and DNA synthesis are in the opposite direction (82). The 11’
protein seems to provide energy for propelling the translocation of the
primosome by hydrolysis of ATP (85). dnaB is proposed to induce a
conformational change in the DNA so that it can be used as a template by

primase (86). dnaC, in a complex with dnaB in the presence of ATP, is

26
pictured at the present time to deliver dnaB to the primosomal complex (87,
88). The functions of protein 11”, n, and i are still unknown.

3. dnaA Protein

Because no readily assayable functions are associated with dnaA protein,
its purification was delayed until the successful establishment of the following
two things: 1) cloning of the dnaA structural gene under control of an
inducible promoter in a high copy number plasmid; 2) development of a crude
enzyme system dependent on dnaA protein that can initiate DNA replication
from the cloned mg in an 911°C plasmid.

The coding sequence of the dnaA gene was not revealed until 1982 (89-
91). The DNA sequence indicated that dnaA encodes a 52.5 KDa protein. At
about the same time, a crude enzyme system that replicates mg plasmids
was developed (92). Although lysates from wild type cells of E. 9211 were
inactive in supporting replication from mg. active protein fractions were
obtained by fractionation of the crude lysate by selective ammonium sulphate
precipitation. These enzyme fractions sustained bidirectional replication of
911°C plasmids from the cloned 9:19 (93). In contrast to the protein fractions
from wild type cells, those from two dnaA mutant strains were inactive.
Addition of a crude lysate from a dnaA protein overproducing strain
complemented the inactive fractions in mg plasmid replication. These
inactive protein fractions from dnaA mutants provided an assay for dnaA
protein. This assay will be referred to as the complementation assay.

27

dnaA protein was then purified in large quantity from an overproducing
strain (94). Two forms of dnaA protein were observed: an aggregated form
eluted in the void volume of a gel permeation column, and a monomeric form.
The reason for the presence of two forms is unknown. Studies to be discussed
are with the monomeric form. dnaA protein was observed to bind to the ma
minimum sequence (95, 96). It also binds to several other sites which will be
discussed later. The binding sites share a consensus sequence
'I'I‘AT(A/C)CA(A/C)A, which is present four times in the 911°C region (95, 96).

4. Mechanism of Initiation from 911°C

Experiments to address the biochemical mechanism of mi!) plasmid
replication were made possible by development of a replication system
dependent on purified enzymes (97). Twelve enzymes were included in the
assay to fulfil the need for several functions in replication: RNA polymerase,
dnaA, DNA gyrase for initiation; DNA polymerase III, primosomal proteins,
and SSB for chain elongation; and topoisomerase I, RN aseH, and protein HU
(a histone like protein) for specificity control. The assay sustained extensive
DNA synthesis of mg plasmids. Replication was dependent on dnaA, dnaB,
dnaC protein, DNA gyrase, and mg. The dependence on RNA polymerase
was more pronounced than that on primase.

Upon varying the amount of topoisomerase I and protein HU, an assay
system completely dependent on primase was established (98, 99). Unlike the
RNA polymerase dependent assay, the specificity of orig-dependent DNA

synthesis does not rely on the presence of RN aseH, indicating that the

28

Figure 4. A model for initiation of replication from 91:19 (106).

29

3.4

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mar—fl
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30
priming was mic specific. To study the initiation process, priming was
uncoupled from DNA replication by omitting DNA polymerase III and dNTPs
from the assay. The priming process which requires dnaA, dnaB, dnaC
protein, DNA gyrase, and primase occurs at an optimum temperature of 36°C
and cannot happen below 24°C. DNA synthesis, on the other hand, can occur
even at 15°C to relatively the same extent as at 30°C. Further dissection of
the priming process indicated that it is not priming itself, but a pre-priming
step which is temperature dependent. The prepriming only requires dnaA,
dnaB, dnaC and DNA gyrase. The presence of SSB also stimulates the
process.

Detailed studies have examined the function of each individual proteins
involved in the prepriming process (Figure 4). dnaA, a sequence-specific DNA
binding protein, recognizes mic and binds to it. Binding induces localized
strand melting at the 13-mer repeat region on the left side of the minimum
sequence (100). This localized unwinding is temperature dependent and
requires the presence of ATP. The involvement of ATP in strand melting is
rather complicated (100, 101). It seems to function as an allosteric factor as
well as an energy source (100). dnaA protein binds ADP as well as ATP. The
ADPdnaA is inactive in strand melting and subsequent replication (100, 101).
However, binding of dnaA protein to gn’Q is not affected by the presence or
absence of either ATP or ADP (101).

Further opening of the 9119 region requires the combined action of dnaB
and DNA gyrase (102, 103). dnaB functions as a helicase to separate the
double-stranded DNA by hydrolyzing ATP. DNA Gyrase releases the localized

31
positive supercoils created by dnaB, which also requires ATP as an energy
source. SSB stabilizes the single-stranded region generated by dnaB protein
and DNA gyrase.

The function of dnaC protein has been considered as delivering dnaB
protein to the dnaA protein-mic complex (87 , 88). dnaC protein forms a very
stable complex with dnaB in the presence of ATP. So far no specific functions
of dnaC have been observed with the prepriming complex. Electron
microscopic studies using gold labeled antibodies failed to locate dnaC protein
in the prepriming complex, although both dnaA and dnaB have been localized
with the same method (105).

Transcription by RNA polymerase seems to assist in strand separation
rather than in primer formation for DNA replication (106). Promoters placed
within 200 bp from the border of the ma sequence but directing transcription
away from the ma also activate DNA replication. However, this does not
exclude the possibility that RNA polymerase primes DNA synthesis, especially
in the absence of primase.

In summary, dnaA protein is required for recognizing 921C and for the
initial unwinding of duplex DNA. Other proteins function in successive steps

of priming and replication (Figure 4).

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32
C. Other Functions of dnaA Protein

1. Involvement in Replication of Plasmids pSC101, R1, R100, F, P1 and ColEl

The dnaA protein recognition sequence 'I'I‘AT(A/C)CA(A/C)A is present at
or near replication origins of plasmids pSClOl, R1, R100, F, P1, and ColE1.
The requirement for dnaA protein is absolute for replication of pSC 101.
Replication is completely blocked in a dnaA46 strain at nonpermissive
temperature (107-109). By contrast, replication of R1, R100, F, and P1 were
once thought not to require dnaA protein for replication since integration of
these plasmids into the chromosome suppressed the temperature sensitive
phenotype of a dnaA46 strain. Studies under more stringent conditions
indicate that these plasmids cannot be maintained in dnaA insertion or
deletion mutants where dnaA protein was completely absent (110-115). The
role of dnaA protein in replication of these plasmids has not‘ been resolved.
dnaA protein is also involved in replication of ColE1 plasmid. Studies with
purified enzymes indicate that dnaA protein directs the assembly of dnaB,
dnaC, and primase, to the replication origin, which is presumably required for
lagging strand synthesis (116, 117). .

2. Riapressor Function in Gene Expression

As early as 1973, Sompayrac and Maaloe proposed that expression of the
initiator protein is controlled by a repressor which also represses the
expression of its own gene (118). This simple feedback control system provides

a constant concentration of the repressor and the initiator. The rate of

33
initiator accumulation should be proportional to the rate of increase in cell
volume. Assuming initiation begins when a certain amount of initiator protein
has accumulated, the frequency of initiation then could be coupled to the rate
of growth.

dnaA protein has been shown to autoregulate its own expression
transcriptionally (119, 120). Two transcriptional promoters separated by 82 bp
were mapped in the dnaA regulatory region (90). One dnaA protein
recognition sequence is present between the two promoters, 51 bp upstream
from the mRNA start site of the proximal promoter, and 23 bp downstream
from the distant promoter. Inactivation of dnaA46 at nonpermissive
temperature resulted in elevated levels of B-galactosidase whose expression is
under control of the two dnaA promoters. High levels of dnaA protein
inhibited expression of B-galactosidase. Detailed examination indicated that
the dnaA protein recognition sequence is required for the repression. Both
promoters seem to be repressed by dnaA protein (121). Autoregulation of
dnaA protein, the initiator, circumvented the need for an additional repressor
in the Sompayrac and Maaloe model.

Other than regulating expression of its own gene, dnaA protein was
observed to repress expression of a gene encoding a 16 KDa protein in vivo
(122, 123). This gene is located to the right of 93°C,. The function of this
protein is still unknown. One dnaA binding site was found about 40 bp
upstream of the mRNA start site. Transcription from the 16 KDa promoter
seems to be involved in the control of initiation (124-126).

34
II. The Heat-shock Response in E. c911

E. c911 can maintain balanced growth over a temperature range from
10°C to 42°C, with optimum temperature at 37°C (127). Temperatures higher
than 42°C result in restricted growth (128). When cultures are shifted to
elevated temperatures, synthesis of more than seventeen proteins, named
heat-shock proteins, is observed to increase transiently (128, 129). This
phenomenon is termed the heat-shock response. Expression of heat-shock
proteins is under control of a regulatory protein, the product of mgH gene
(130, 131). The moH, gene encodes a sigma factor of 32 KDa which confers
upon RNA polymerase the ability to initiate transcription from promoters of
heat-shock genes (132, 133). Stress conditions other than heat, such as
ethanol in the culture media, UV irradiation, and chemicals that block DNA
synthesis or change DNA structure, are also known to trigger the heat-shock

response (128).

A. Heat-shock Proteins

Eight heat-shock proteins have been identified since the discovery of the
heat-shock response in E. £911 in 1978 (129). These proteins are involved in
diverse cellular processes from DNA replication, RNA synthesis, to protein
processing.

Some heat-shock proteins are among the most abundant proteins of E.
can. The product of groEL gene is one of them, which constitutes about 1-2%
of the total cellular protein at 37°C (128). The 65 KDa groEL protein has

35
been purified and it exists as homopolymeric particles (134, 135). It
functionally interacts with another heat-shock protein, the 15 KDa groES
protein (136, 137). The ng genes were first defined by mutations affecting
the morphogenesis of several bacterial phages (137, 138). Recently, the groE
proteins are shown to be associated with newly synthesized peptides and to
assist in assembly of multimeric enzyme complexes (139-141). The groE
proteins are required for growth at all temperatures (142).

dnaK protein is another abundant heat-shock protein (143). It possesses
ATPase and phosphorylase activities (144). dnaK functions with dnaJ, another
heat-shock protein in replication of lambda phage (145). Mutations in M or
final also inhibit cellular DNA replication and RNA synthesis (146, 147, 148).

grpE, also involved in lambda replication, is a member of heat-shock
proteins (149). Like dnaK and dual, mutations in ng also cause reduced
synthesis of both RNA and DNA ( 149). Detailed fimctions of grpE protein are
unknown.

Protease La, an ATP dependent protease encoded by Inn, is the sixth
member of the heat-shock protein family (150-151). It provides a major route
by which E. mli degrades abnormal proteins (153, 154). The last two
members of the heat-shock proteins known so far are the sigma 70 subunit of
RNA polymerase and the 12311 gene product, a minor form of lysyl-tRNA
synthetase (128).

36
B. The Heat-shock Regulatory Protein, Sigma32

Expression of heat-shock proteins is controlled by the product of anti
gene, a 32 KDa sigma factor of RNA polymerase (130-133). Sigma32 RNA
polymerase recognizes specific patterns of -10 and -35 regions characteristic of
heat-shock promoters (155).

The level of sigma32 increases transiently up to 15 fold after a
temperature shift (156, 157). The transient accumulation of sigma32 is a
result of both increased synthesis and increased stability. A temperature shifl:
from 30° to 42°C has been shown to lengthen the half life of sigma32 from
about 1 minute to 8 minutes within the first 4 minutes following the shift.
Thereafter, the half life returns to about 1 minute, which causes a relative
decrease in the level of sigma32 (157, 158). The amount of sigma32, as well
as levels of the heat-shock proteins, then stabilizes at a level characteristic of
the growth temperature.

The regulation of sigma32 has been examined at transcriptional level
(159-161). Four promoters has been mapped in the regulatory region,
designated mHlP, 2P, 3P, and 4P, with 4P being the most proximal. 2P is
only observed with one specific strain; other promoters are observed both in
vivo and in vitro. Transcripts from 1P are most abundant at all temperatures.
Its level increases moderately (2 fold from 30°C to 42°C) with temperature.
Transcripts of 3P and 4P are present at low levels at 30°C, and increase 5-15
fold transiently after heat shock. This increase correlates well with rate of
synthesis of sigma32. The presence of high levels of 1P RNA even at 30°C

where levels of sigma32 are extremely low suggests posttranscriptional

37
regulation on expression of the 1P transcript.

C. Regulation of the Heat-shock Response

Current evidence indicates that dnaK protein plays an important role in
the control of the heat-shock response. Mutants of dnaK show increased levels
of heat-shock proteins and prolonged synthesis of these proteins at high levels
after heat shock (162, 163). These results indicate a negative regulatory
function of dnaK protein.

The level of ppGpp, the messenger of stringent response when amino acid
is limiting, has been observed to increase transiently after heat shock in a
similar pattern as that of sigma32 and of heat-shock proteins (166, 167). High
levels of ppGpp are associated with mutations in dnaK and dnaJ (148). M
mutants also fail to phosphorylate at least five proteins. Two of them are
identified as glutaminyl-tRN A synthetase and threonyl-tRN A synthetase (164).
dnaJ protein also participates in the phosphorylation of the latter two. The
accumulation of ppGpp seems to be a result rather than a cause of the
absence of phosphorylation of the tRNA synthetases in dnaK and dnaJ
mutants (164). Related to these observations is the finding that stringent
response induces expression of heat shock proteins (165). These results
indicate a direct correlation among dnaK (dnaJ), the level of ppGpp, and
synthesis of heat shock proteins.

A model is formulated from the above evidence. 1. A temperature shift
might result in elevated levels of ppGpp through the following connection.

Glutamate and threonine are precursors of many other amino acids (168). A

38
sudden increase in temperature might result in an increase in demand for
these two amino acid in several biochemical pathways. This, in turn, may
cause a temporary shortage of glutamate and threonine. High level of ppGpp
could then produced as a result. 2. Synthesis of heat shock proteins could be
stimulated by ppGpp (165). 3. Production of dnaK and dnaJ protein might
modulate the response. High levels of dnaK and dnaJ protein, resulting from
the elevated synthesis of heat shock proteins, might modulate the activities of
glutaminyl-tRNA and threonyl-tRNA synthetase by phosphorylation, which
presumably could result in decreased synthesis of ppGpp. The levels of
ppGpp, sigma32, and the rate of synthesis of heat shock proteins will
decrease until reaching a new balance.

III. Objectives of the Project

dnaA protein recognition sequences are present not only in mg, the
dnaA and 16 KDa promoter regions but also in promoter regions of several
other genes, including mgH, pom. uvrB, and and. This project was
undertaken to determine if dnaA protein regulates expression of these and
other genes. Further understanding of dnaA protein as a regulatory protein
will help to correlate DNA replication with other cellular processes during cell
growth.

10.

11.

.13.
14.
15.
16.
17.

18.
19.

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Chapter II
TRANSCRIPTIONAL REPRESSION OF THE dnaA GENE
OF Esghgfimm c911 BY dnaA PROTEIN

49

ABSTRACT

The promoter regions of the dnaA gene and of a gene which encodes a
16KDa protein contain sites which are recognized and bound by dnaA protein.
Using assays of run-off transcription of restriction fragments, purified dnaA
protein specifically repressed transcription from both dnaA promoters and
from the promoter for the 16 KDa gene to almost undetectable levels. This
repressive effect is observed at levels of dnaA protein required for specific
binding of dnaA protein to restriction fragments containing the promoters for
these genes. These results indicate that transcription of these genes is
regulated by binding of dnaA protein to the promoter regions of these genes.

50

INTRODUCTION

The dnaA gene of E. cgh is an essential gene involved in the initiation of
DNA replication from the chromosomal origin, mg. Genetic, physiological,
and biochemical experiments indicate that the dnaA product acts at an early
stage in initiation of DNA replication (21, 41, 28, 15). Its mechanism of action
in initiation of replication remains to be completely clarified. Various results
have indicated that this initiator protein may play a central role in the
regulation of chromosomal replication. Although temperature sensitive
mutants of dnaA fail to initiate new rounds of DNA synthesis when shifted to
the nonpermissive temperature, some mutants of dnaA accumulate excess
initiation capacity at the nonpermissive temperature (18, 11, 39, 29). When
these strains are returned to the permissive temperature, new initiations
occur in the absence of protein synthesis. The capacity to initiate DNA
replication under such conditions has led to the suggestion that the dnaA
product regulates the initiation of DNA replication and also represses its own
expression (18, 26, 12). Evidence supporting this is that overproduction of
dnaA protein, when under control of the lambdaPL or lagUV5 promoters,
resulted in an increased frequency of initiation of DNA replication (2, 6).

The dnaA gene is the first gene in an operon also containing the dnaK
gene which encodes the 6 subunit of DNA polymerase III holoenzyme (34, 8).
The dnaA gene has been cloned and its DNA sequence determined (19, 33, 17,

32). S1 nuclease mapping experiments of transcripts produced in viva indicate

51

52
that two promoters, dnaAlP and dnaA2P located approximately 230 bp and
150 bp upstream from the translational start site, are responsible for
transcription of the dnaA gene (20, 4).

Using assays of in vitro replication of plasmids containing the
chromosomal origin of E. £011 in conjunction with overproduction of the dnaA
gene product, highly purified preparations of dnaA protein have been obtained
(14). Characterization of such preparations has indicated that dnaA protein is
a sequence-specific DNA binding protein (10, 13). DNA fragments bound by
dnaA protein include those containing the ma sequence, the promoter region
of a “gene adjacent to uric which encodes a 16 KDa protein, and the dnaA
promoter region. From comparative sequence analysis and DNase I
footprinting experiments, it is postulated that dnaA protein recognizes a
specific 9 base pair sequence in binding to these DNA fragments (13).

Recent studies have examined the in viva expression of the dnaA gene by
using transcriptional and translational gene fusions of the dnaA promoter
region to the structural region of the lacZ gene (4, 2) or by S1 nuclease
mapping (27 ). Over-production of the dnaA gene product led to decreased
expression from the dnaA promoters. In contrast, introduction of the dnaA46
allele resulted in elevated expression from the dnaA promoters at the
nonpermissive temperature. In the former studies, deletion of one of the two
dnaA promoters (dnaAlP) and the dnaA protein recognition sequence resulted
in the loss of this autoregulatary efi‘ect. These results suggest that dnaA
protein autoregulates its own expression by recognizing and binding to the
dnaA promoter region. The inability of the dnaA46 gene product to repress

53
expression of B-galactosidase activity when under control of the dnaA
regulatory region is presumably due to its inability to bind to this region.

Transcriptional repression of a gene adjacent to mg by dnaA protein has
also been observed by use of gene fusions of the 16 KDa promoter to the galK
gene (30, 38). Expression of galactokinase in vivo or in coupled
transcription/translation assays was dependent on the promoter region of the
16 KDa gene.

A biochemical understanding of the binding of dnaA protein to the
chromosomal origin, to its promoter region, and to promoter regions of other
genes in relation to its effects on transcription may contribute to the
understanding of the regulation of initiation. The results presented in this
report indicate that purified dnaA protein functions to specifically repress
transcription of the dnaA gene and of the gene encoding a 16 KDa protein.
This inhibition is observed at levels which correspond to those required for
binding of dnaA protein to DNA fragments containing the promoter region of

these genes.

MATERIALS AND METHODS

W

The plasmid pdnaA/dnaN is a recombinant of pMOB45 which contains
the dnaA and dnaK genes within the inserted 1.0 and 3.3 kilobase pair
153le fragments (8). Plasmid pBF1509 contains the E. 9.011 chromosomal DNA
fragment of pBF1209 (13) inserted into the vector pAD329 (23). This DNA
fi'agment was modified near the ClaI site by attachment of a BamH1 linker.
These plasmids, M13QriQZ6 RF (25), and pBR322 were purified from cleared
lysates by centrifugation in ethidium bromide-CsCl gradients. The 203 bp
restriction fragment containing the lagUV5 promoter was a gift of D. Lorimer
of this department.

Restriction enzyme digests for filter binding assays and for purification of
restriction fragments were performed according to the manufacturers’
recommendations. Restriction fragments for run-off transcription assays were
fractionated on polyacrylamide gels, visualised by ethidium bromide staining,
purified by electroelution with an 1800 Model 1750 electrophoretic
concentrator, and ethanol precipitated. DNA concentrations were determined
spectrophotometrically, or by comparison of the purified DNA fragments to
different known amounts of electrophoretically separated restriction enzyme
digests visualized by ethidium bromide staining.

54

55

Enzymes

Restriction enzymes EcoRI, BamHI, and Tan were purchased from New
England Biolabs, HaeIII and Hinfl were from IBI, and HpaII was from
Boehringer/Mannheim. DNA polymerase I (large fragment) was fi'om New
England Biolabs. RNA polymerase holoenzyme was purified from W3110 as
described except that Biogel A5m (Biorad) chromatography was replaced by
chromatography on a TSK 3000SW (Altex) high performance gel permeation
column (9, 16). dnaA protein was purified as described (24). Protein
concentrations were determined by the dye-binding method with bovine serum
albumin as a standard (3).

DH! 1 . l'

Fragment retention assays were performed using Millipore HAWP
nitrocellulose filters (13). Filters, boiled (5 min), and stored in H,O at 4°C,
were equilibrated in binding buffer containing 40 mM HEPES-KOH pH 7.6, 5
mM magnesium acetate, 2 mM dithiothreitol, and 50 mM KCl prior to use.
Binding assays (25 pl) were performed in the above buffer containing 0.025
pmol of purified restriction fragment digested with the indicated restriction
enzyme and end-labeled with DNA polymerase I (large fragment) (40). Various
amounts of dnaA protein were added in 1 111 of dnaA bufi'er containing 25
mM HEPES-KOH pH 7.6, 15% glycerol, 0.1 mM EDTA, 2 mM dithiothreitol,
and 0.1 M (N11,),SO,. Reactions, incubated for 10 min at 30°C, were
immediately filtered under gentle suction and the filters were washed with
250 111 of binding buffer. DNA retained on the filters was eluted in buffer

56
containing 25 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.5 M sodium acetate and
0.5% sodium dodecyl sulfate at 65°C for 20 min. DNA eluted from the filters
and in the flow through were concentrated by ethanol precipitation with 10
ug of E. c911 tRNA (Sigma) as carrier, dissolved in buffer containing 5.0%
glycerol, 0.1% bromophenol blue, 10 mM Tris-HCl pH 8, and 1 mM EDTA,
and electrophoresed in 1.5% agarose gels in 80 mM Tris-borate pH 8.3, and 1
mM EDTA. After electrophoresis, the gels were stained with ethidium
bromide, photographed, dried onto Whatman DE81 paper and
autoradiographed at -7 0°C with Kodak XAR-5 film and Cronex Quanta III
intensifying screens.

Wm

Run-ofi‘ transcription assays (10 pl) contained 0.025 pmol of the indicated
restriction fragment, 0.65 pmol of RNA polymerase, and varying amounts of
dnaA protein in 1 ul of dnaA buffer in 40 mM Tris-HCl pH 7.8, 10 mM
MgCL, 0.1 mM EDTA, 0.1 M NaCl, 1 mM dithiothreitol, 10% glycerol, 0.1
mg/ml bovine serum albumin, 300 M each of ATP, GTP, and CTP, and 20
11M [a‘p] UTP (5). Incubations of 10 min. at 37°C were terminated by
addition of an equal volume of stop buffer (0.1% SDS, 20 mM EDTA, 0.3 M
sodium acetate, and 100 11ng E. 5:911 tRNA). Reaction products were ethanol
precipitated, resuspended in 20 ul of 95% formamide, and 40 mM Tris-HCl
pH 7.8, incubated at 100°C for 2 min., chilled on ice, and electrophoresed in
5 or 8% polyacrylamide gels in 7 M urea, 90 mM Tris-borate pH 8.3, and 1
mM EDTA. pBR322 DNA digested with EcoRI and HpaII and end-labeled

57
with DNA polymerase I (large fragment) was used as a molecular weight
marker. After electrophoresis, gels were soaked for 10 min. in H,O before
drying and autoradiography as described above. Autoradiograms were
quantitated by densitometry with a Hoefer gel scanner interfaced with an
IBM personal computer.

R l' |° ll 11' [ENE

Restriction fragments (1-5 ug) for DNA-binding experiments were
end-labeled with 2 uCi of [OPP] dCTP (New England Nuclear, 800 Ci/mmol)
and one unit of of DNA polymerase I (large fragment) in 10 ul of 10 mM
Tris-H01 pH 7.3, 10 mM MgCl,, and 50 mM NaCl (HpaII digested DNA) or
100 mM NaCl (Tan digested DNA) for 20 min at room temperature. The
end-labeled restriction fragments (0.5-1 x 10‘ cpm/ug of DNA by acid insoluble
radioactivity) were used directly in filter binding experiments.

RESULTS

00:; 00'! iO‘flii WWHOOOO It“ O'Otié Outfittcl

In fragment retention assays and DNase I protection experiments,
purified dnaA protein has been shown to bind in a cooperative fashion to a
region of 100 bp containing the dnaA promoters and to the promoter region
for a gene encoding a 16 KDa protein (Figures 1.2) (13). Levels of dnaA
protein sufficient for specific binding and for protection correspond to a ratio
of approximately 50 monomers of this 52 KDa protein per bound DNA
fragment.

An EcoRI restriction fragment of 945 bp was isolated from a recombinant
of pMOB45 (8) which contains the promoters required for divergent
transcription of the anti and dnaA genes (20, 32, 4). Addition of RNA
polymerase to run-ofi‘ transcription assays containing this restriction fragment
as template resulted in the synthesis of several products (Figure 3a). Two
transcripts of approximately 500 and 380 nucleotides were observed which
correspond in size to those products expected by transcription from mmHlP
and mmH2P (505 and 378 nucleotides, respectively) (20). Two other
transcripts of approximately 300 and 215 nucleotides were observed which are
consistent in length with transcription fi‘om dnaAlP and W (20, 4). A
transcript of 130 nucleotides was also observed which may result from
transcriptional termination from mmH1P (20). The largest product is most
likely due to end to end transcription of the restriction fi'agment since its size

58

59

Figure 1. Map of the dnaA and 1211111 promoter regions and coding regions
contained in the 945 bp EcoRI fragment (20).

Restriction sites: E, EcoRI; Hf, Hinfl; Hp, HpaII; H, HaeIII; C, ClaI. The
approximate positions of the promoters for the dnaA gene, dnaAlP and
MP the), and for the rpmH gene, mmHlP and mmHZP (<-«C=D are
indicated. The direction of transcription is indicated by the arrows. The
stippled box indicates the position of the dnaA protein recognition sequence;
cross hatched boxes indicates open reading frames for known gene products.
The dashed box indicates an open reading frame for a putative protein.
Restriction fragments used in run-off transcription assays are indicated.

60

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Figure 2. Map of the gene encoding the 16 KDa protein and adjacent aan
gene (22, 42).

Restriction sites: T, Tan; Hp, HpaII. The approximate positions of the
promoters for the gene encoding the 16 KDa protein (<--::3, and for the
adjacent aan gene (<--l::), and the direction of transcription are indicated.
The restriction fragment used in run-off transcription assays is also indicated.

Other designations are as in Figure 1.

62

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63

Figure 3. Run-off transcription using the 945 bp EcoRI restriction fragment.
(A) Assays were performed as described in Materials and Methods with
the indicated amounts of dnaA protein. The approximate sizes of the
transcripts were determined relative to EcoRI, HpaII digested pBR322 DNA
as a size standard (left column).
(B) The amounts of each transcript determined as described in Materials

and Methods are expressed as a ratio compared to the control with no dnaA
protein added.

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is approximately equal to the length of the fragment. Similar products
attributed to transcription from dnaAlP and dnaA2P have been observed by
others in run-off transcription assays with preparations of the same EcoRI
restriction fragment (32, 5).

Addition of various amounts of purified dnaA protein to run-ofi‘
transcription assays containing this EcoRI fragment resulted in a preferential
inhibition of synthesis of both 300 and 215 nucleotide transcripts (Figure
3A,B). This result is consistent with inhibition from dnaAlP and WP by
dnaA protein. At the highest level of dnaA protein added, synthesis of the 300
and 215 nucleotide transcripts was inhibited about 10 fold (Figure 3B). The
amount of dnaA protein sumcient for maximal inhibition of transcription is
comparable to that amount required for preferential binding of dnaA protein
to restriction fragments containing the dnaA promoter region (13, see below).
In contrast, synthesis of transcripts of 380 and 130 nucleotides was inhibited
by less than 2 fold at this level. It was not possible to quantitate accurately
the amount of the 500 nucleotide transcript due to high backgron in this
region of the autoradiogram.

Cleavage of this 945 bp EcoRI fragment with HaeIII restriction enzyme
removes the mmHZP promoter and coding sequences distal to and
downstream from the M promoters (figure 1). Run-ofl' transcription assays
with this 569 bp restriction fragment as a template resulted in transcripts of
approximately 300 and 215 nucleotides (Figure 4A) which are similar in size
to those observed with the larger 945 bp restriction fragment as a template
(Figure 3A). In addition, two transcripts close to 130 nucleotides in length

66

Figure 4. Run-off transcription using the 569 bp HaeIII-EcoRI restriction
fragment.

(A) Reactions were performed as described in Materials and Methods with
the indicated amounts of dnaA protein. The sizes of the transcripts were
determined relative to EcoRI, HpaII digested pBR322 DNA as a size standard
(left column).

(B) The amounts of each transcript determined as described in Materials
and Methods are expressed as a ratio compared to the amount of transcript

produced in the absence of dnaA protein.

67

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were observed while products of 500 and 380 nucleotides were absent. The
appearance of one of these transcripts with this shortened restriction
fragment is consistent with the termination product observed in Figure 3A
formed by transcription from mmHlP. The slightly longer transcript is
consistent with run-off transcription from mmHlP. Based on size, the largest
product is presumed to result from end to end transcription of the restriction
fragment. The addition of increased amounts of dnaA protein to reactions
containing RNA polymerase and this restriction fragment resulted in up to a
twenty-fold repression of synthesis of the 300 and 215 nucleotide transcripts.
Marginal inhibition by dnaA protein was observed with the 130 nucleotide
transcript (Figure 4B). Levels of dnaA protein sumcient for maximal and
preferential inhibition of synthesis of the 300 and 215 nucleotide transcripts
were similar to that of Figure 3.

Digestion of pBR322 with EcoRI and HpaII restriction enzymes produces
a 459 bp restriction fragment containing the promoter and N-terminal coding
region of the B—lactamase gene. This DNA fragment is poorly bound by dnaA
protein and contains no striking homologies to the dnaA protein recognition
sequence (13). Other studies which have determined the location of the
promoter and transcriptional start site for this gene predict a 288 nucleotide
run-ofl' product upon transcription of this restriction fragment (7). The
observed product of about 290 nucleotides is in agreement with the expected
size (Figure 5A). Addition of dnaA protein to run-ofl' transcription assays
containing this restriction fragment resulted in a modest level of inhibition of
transcription from the B—lactamase promoter (Figure 5A, C). The modest level

69

Figure 5. Run-off transcription from the B-lactamase promoter and from the
lacUV5 promoter.

(A) Assays were performed with the 459 bp EcoRI-HpaII restriction
fragment containing the B—lactamase promote.

(B) Run-ofl‘ transcription assayed with the 203 bp restriction fragment
containing the lacUV5 promoter. Sizes of transcripts were determined relative
to EcoRI, HpaII digested pBR322 DNA as a size standard (left column).

(C) The amounts of each transcript determined as described in Materials
and Methods are expressed as a ratio compared to the amount of transcript
produced in the absence of dnaA protein.

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of inhibition observed is similar to the effect observed for transcription from
mmHlP and mmHZP (Figure 3,4). As above, the largest transcript can be
attributed to end to end transcription of the restriction fragment. A transcript
of about 190 nucleotides was also observed. The presence of this transcript is
unexpected.

Run-off transcription experiments were also performed with a 203 bp
restriction fragment containing the lagUV5 promoter. By sequence analysis,
this DNA fragment lacks sequences homologous to the consensus dnaA
protein recognition sequence and is poorly bound by dnaA protein (see below).
The observed transcripts of 60-70 nucleotides are similar to those previously
observed by transcription from the lacUV5 promoter with RNA polymerase in
the absence of cyclic AMP binding protein (Figure 5B) (31). The addition of
increasing amounts of dnaA protein to these assays minimally afl‘ected on
synthesis of these transcripts (Figure 5B, C). The product of 205-210
nucleotides is prsumably due to end-to-end transcription of the restriction
fragment. The product of about 280 nucleotides was unexplained but has been
observed by others in transcription of the same fragment containing the
lacUV5 promoter (43).

Construction of pBF1509 results in a 397 bp BamHI-EcoRI fragment
which separates the dnaA promoter region from the mmH promoter region
(13). Run-ofi' transcription with this restriction fragment resulted in synthesis
of 215 and 300 nucleotide transcripts (data not shown). Addition of dnaA
protein inhibited synthesis of both transcripts to almost undetectable levels
(data not shown). The 397 bp BamHI-EcoRI fragment containing the dnaA

72
promoter region was digested with Hian which removes additional coding
sequences of the dnaA gene. If the 215 and 300 nucleotide products observed
above were due to transcription from dnaAlP and dnaA2P, transcripts of 231
and 146 nucleotides are expected with this 328 bp BamHI-Hinfl fragment. As
indicated in Figure 6A, transcripts of approximately 230 and 140 nucleotides
were observed. The addition of dnaA protein in increasing amounts resulted
in a corresponding decrease in synthesis of both of these transcripts to
undetectable levels (Figure 6B). The largest product corresponds in size to end
to end transcription of the restriction fragment. A transcript of about 70
nucleotides was also observed. Although the appearance of this transcript is
unexplained, its synthesis is not inhibited by the addition of dnaA protein.
Parallel reactions were performed of run-off transcription fiom the lacUV5
promoter and the B—lactamase promoter. These transcripts were marginally
inhibited upon addition of similar levels of dnaA protein (data not shown).

0“; cu -. :own : : amazwo u mu o- . JD: ouuo -
Experiments to determine the influence of dnaA protein on transcription
of the 16 KDa gene adjacent to am: were performed. The promoter region for
this gene encoding a 16 KDa protein is contained in a 414 bp HpaII
restriction fragment (22). Run-ofl‘ transcription assays with this restriction
fragment resulted in formation of a 200 nucleotide transcript which was
inhibited up to 20-fold upon addition of increasing amounts of dnaA protein
(Figure 7A,B). As in Figure 5, addition of similar amounts of dnaA protein to

parallel run-off transcription assays with a restriction fragment containing

73

Figure 6. Run-off transcription using the 328 bp BamHI-Hinfl restriction
fragment.

(A) Assays were performed as described in Materials and Methods with
the indicated amounts of dnaA protein. The sizes of transcripts were
determined relative to EcoRI, HpaII digested pBR322 DNA as a size standard
(left column).

(B) The amounts of each transcript determined as described in Materials
and Methods are expressed as a ratio compared to the amount of transcript

produced in the absence of dnaA protein.

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Figure 7. In vitro transcription using the 414 bp HpaII restriction fragment
containing the promoter region for the 16 KDa protein.

(A) Assays were performed as described in Materials and Methods with
the indicated amounts of dnaA protein. The approximate sizes of transcripts
are indicated.

(B) The amounts of each transcript determined as described in Materials
and Methods are expressed as a ratio compared to the control with no dnaA
protein added.

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the lagUV5 promoter resulted in less than 2-fold inhibition of transcription
(data not shown).

$151.1. E13 |.| |’|'fi |
Fragment retention assays were performed with dnaA protein and DNA

fragments used in run-ofl' transcription assays to determine whether inhibition
of transcription by dnaA protein correlated with its ability to bind to
restriction fragments containing dnaA protein recognition sequences.

The 945 bp EcoRI fragment was digested with HpaII restriction
endonuclease and 3’ end-labeled with the large fragment of DNA polymerase I
and aaP-dCTP. After incubation with various amounts of dnaA protein,
fragments which bound to or flowed through nitrocellulose filters were
visualized after gel electrophoresis and autoradiography (Figure 8A). The
largest fragment of 445 bp contains the dnaA protein recognition sequence
between dnaAlP and dnaA2P and the promoter, mmHlP. This fragment was
more tightly bound by dnaA protein than the 37 8 bp fragment containing the
:pmH and mpA genes (Figure 8B). The 95 bp and 21 bp fragment between
mmHlP and WP was poorly bound under these reaction conditions.

Fragment retention experiments were also performed with the 414 bp
restriction fragment containing the promoter region for the 16 KDa gene
combined with an equimolar amount of the 203 bp restriction fragment
containing the lacUV5 promoter. The addition of dnaA protein resulted in
preferential retention of the 16 KDa promoter-containing fragment compared
to binding to the lacUV5 promoter fragment (Figure 9A,B). The amount of

78
dnaA protein required for preferential binding to restriction fragments
containing the promoters for the dnaA or 16 KDa genes corresponded to the
amount of dnaA protein required for specific inhibition of transcription from

these promoters.

79

Figure 8. Preferential binding of dnaA protein to a restriction containing the
dnaA promoters.

Fragment retention assays were performed as described in Materials and
Methods with 0.025 pmol of the 945 bp EcoRI fragment digested with HpaII
and the indicated amounts of dnaA protein.

(A) Positions and sizes of the subfragments produced by HpaII digestion
are indicated. TD, total digest; B, bound fragments eluted from filters; FI‘,
fragments which flowed through the filter.

(B) Amounts of fragments bound were determined as described in
Materials and Methods.

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Figure 9. Preferential binding of dnaA protein to a restriction fragment
containing the 16 KDa promoter.

Fragment retention assays were performed as described in Materials and
Methods with 0.025 pmol each of the 414 bp fragment containing the 16 KDa
promoter and of the 203 bp fragment containing the lacUV5 promoter.

(A) Positions and sizes of the restriction fragments are indicated. TD, B,
and FT are as in Figure 8.

(B) Amounts of fragments bound were determined as described in
Materials and Methods.

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DISCUSSION

In the studies reported here, transcriptional repression of the dnaA gene
by dnaA protein was observed at levels similar to those required for specific
binding of dnaA protein to DNA fragments containing the dnaA promoter
region. Inhibition of transcription by dnaA was observed to affect both dnaA
promoters. Under most reaction conditions, 100 ng of dnaA protein resulted in
about 10 fold inhibition of both of the dnaA promoters. Transcriptional
inhibition of the 16 KDa gene by dnaA protein was also observed at levels
similar to those required for specific binding to DNA fragments containing the
promoter region for this gene. dnaA protein did not markedly inhibit
transcription from the promoter for either the B—lactamase gene, or the
lacUV5 promoter in separate run-ofl‘ transcription assays, or from the mmH
promoters contained on the same restriction fragment as the dnaA promoters.
These results indicate that inhibition is specific. Recognition of the dnaA
protein recognition sequence and binding are directly related to its repressive
effect.

The transcripts observed in these experiments confirm the positions of
the promoters for the dnaA, mmH, lacUV5, and B—lactamase genes ( 19, 22,
31, 7). In one experiment, transcription of the BamI-II-Hinfl fragment resulted
in an unexpected product of 70 nucleotides. This transcript remains
unexplained as larger restriction fragments do not direct the synthesis of a
corresponding transcript. A transcription experiment of a DNA fragment

83

84
containing the B-lactamase promoter resulted in synthesis of a 190 nucleotide
transcript. This product was observed in addition to transcripts attributed to
initiation from the B-lactamase promoter and end-to—end transcription. Based
on $1 nuclease mapping experiments and electron microscopic mapping of
RNA polymerase-promoter complexes, this restriction fragment only contains
one of the two promoters involved in transcription of the B—lactamase gene of
pBR322 (7, 36). This transcript may have arisen by premature termination
from the B—lactamase promoter in this DNA fragment. A termination product
of 190 nucleotides would not have been observed in these studies.

Recent studies relying on transcriptional and translational fusions of the
dnaA promoter region to the 139.2 gene have indicated that overproduction of
the dnaA gene product resulted in decreased expression of B-galactosidase
activity while introduction of the dnaA46 allele resulted in elevated expression
at the nonpermissive temperature (4, 1). Deletion of sequences upstream from
dnaA2P and including dnaAlP resulted in the lack of this repressive efl‘ect.
This deletion also removes the dnaA protein recognition sequence between
dnaAlP and dnaA2P involved in binding of dnaA protein to this region of
DNA.

Other studies in which the promoter for the 16 KDa gene was linked to
the galK structural gene have indicated that galactokinase activity was
decreased with elevated levels of dnaA protein (30, 38). No influence of dnaA
protein on transcription from the 16 KDa promoter was observed with
recombinants lacking 6 bp of the dnaA protein recognition sequence (38). The
results from these in vivo studies suggest that binding of dnaA protein to the

prc

Us
a]

is

ele

be'

CO]

PI"

pr:
ide

85
promoter regions regulates expression of these genes.

The studies presented here are in agreement with these in vivo studies.
Using purified components, maximal inhibition of transcription is observed at
a ratio of 50 to 100 monomers of dnaA protein per recognition site. This level
is consistent with its cooperative binding to the dnaA and 16 KDa promoter
regions, and with the sizes of dnaA protein-DNA complexes observed by
electron microscopy (13). The dnaA protein recognition sequence is located
between the two promoters involved in transcription of the dnaA gene (13). In
comparison, this recognition sequence is located slightly upstream from the
~35 region of the 16 KDa promoter (38). These results suggest that
transcriptional inhibition may involve occlusion of RNA polymerase from
promoters bound by dnaA protein. If dnaA protein interacts directly with
RNA polymerase to influence its activity at promoters containing dnaA
protein recognition sequences, this interaction does not dramatically influence
transcription fi'om the promoters for B-lactamase, mmH, or lacZ.

Experiments of fragment retention on nitrocellulose filters showed that
dnaA protein was weakly bound to the DNA fi‘agment containing the lagUV5
promoter and to the fi'agment containing the M coding region. The result
that transcription of these DNAs was slightly inhibited by dnaA protein may
be due to the ability of nonspecifically bound dnaA protein to inhibit
transcription.

Expression of the dnaA gene involves transcriptional initiation from two
promoters both in vivo and in vitro. Promoters utilized in vitro appear to be
identical to those recognized in the cell (20, 4). Whereas transcription in vivo

86
is reported to occur predominantly from the dnaA2P promoter, the in vitro
studies reported here indicate that both dnaAlP and dnaA2P promoters are
utilized with about equal efi‘iciencies at an RNA polymerasezDNA template
ratio of 26:1. Varying the ratio of RNA polymerase holoenzyme to the DNA
restriction fragment appeared to influence the efliciency of promoter usage
(data not shown). At a 1:1 ratio of RNA polymerase to DNA template,
dnaAZP appeared to be more efliciently utilized. Other factors may modulate
the usage of the dnaA promoters. The observation that dnaA protein binds
with higher afl‘inity to supercoiled than linearized mg plasmid DNA (14)
suggests that the superhelical state of the template may influence the
regulatory effect of dnaA protein on expression of its gene.

The levels of dnaA protein required for autoregulation in vitro are
approximately equal to the levels of dnaA protein required for initiation of
replication on 9:19 plasmid DNAs (15). It has been shown that the rate of
DNA replication is strictly coordinated to the growth rate of the bacterium
such that the ratio of DNA content to cell mass remains constant (35). An
autorepressor model for control of DNA replication was proposed (37). This
model proposes that an initiator protein is contained in an operon which also
encodes a gene product that autoregulates expression of the operon. The
activities of dnaA protein are consistent with its role as an initiator of
replication and as an autoregulator of its expression. From genetic and
biochemical studies, dnaA protein appears to play a central role in initiation
of replication (21, 41, 28, 13). Experiments in which expression of dnaA

protein is elevated by overproduction resulted in an increased frequency of

87
initiation (2, 6). That these new initiations are aborted in the former studies
indicates that the coordination of DNA replication and cell growth is
interrupted or that other factors may become rate limiting.

Whether the dnaA gene product also functions to coordinate DNA
replication to cell growth is unknown. The observation that expression of the
16 KDa gene is negatively regulated by the dnaA gene product and the
presence of sequences homologous to the concensus dnaA protein recognition
sequence in promoter regions for other genes suggests that dnaA protein may
act to influence expression of these genes. The influence of dnaA protein on
expression of these genes can be measured directly with the availability of

cloned DNA fragments containing these genes of interest.

<9:ng

10.

11.

13.
14.

15.

16.

17.

REFERENCES
Atlung, T., Clausen, E., and Hansen, F. G. (1985) Mol. Gen. Genet.
200, 442-450.
Atlung, T., Rasmussen, K. V., Clausen, E., and Hansen, F. G. (1985) in
Molecular biology of bacterial growth. Jones and Bartlett Publ. Inc.,
Portola Valley, CA:p 282-297.
Bradford, M. M. (1976) Anal. Biochem. 72, 248-254.
Braun, R. E., O’Day, K., and Wright, A. (1985) Cell 40, 159-169.
Braun, R. E., and Wright, A. (1986) Mol. Gen. Genet. 202, 246-250.
Bremer, H., and Churchward, G. (1985) J. Bacteriol. 164, 922-924.

Brosius, J., Cate, R. L., and Perlmutter, P. A. (1982) J. Biol. Chem.
257, 9205-9210.

Burgers, P. M. J., Kornberg, A., and Sakakibara, Y. (1981) Proc. Natl.
Acad. Sci. USA 78, 5391-5395.

Burgess, R. R., and Jendrisak, J. J. (1975) Biochemistry 14, 4634-4638.

Chakraborty, T., Yoshinaga, K., Lother, H., and Messer, W. (1982)
EMBO J. 1, 1545-1549.

Fralick, J. A. (1978) J. Mol. Biol. 122, 271-286.

Frey, J., Chandler, M., and Caro, L. (1981) Mol. Gen. Genet. 182,
364-366.

Fuller, R. S., Funnell, B. E., and Kornberg, A. (1984) Cell 38, 889-900.

Fuller, R. S., and Kornberg, A. (1983) Proc. Natl. Acad. Sci. USA 80,
5817-5821.

Funnell, B. E., Baker, T. A., and Kornberg, A. (1986) J. Biol. Chem.
261, 5616-5624. '

Gonzalez, N., Wiggs, J., and Chamberlin, M. J. (1977) Arch. Biochem.
Biophys. 182, 404-408.

Hansen, E. B., Hansen, F. G., and von Meyenburg, K. (1982) Nucleic
Acids Res. 10, 7373-7385.

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18.

19.

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33.

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Hansen, F. G., and Rasmussen, K. V. (1977) Mol. Gen. Genet
155:219-255.

Hansen, F. G., and van Meyenburg, K. (1979) Mol. Gen. Genet. 175,
135-144.

Hansen, F. G., Hansen, E. B., and Atlung, T. (1982) EMBO J. 1,
1043-1048.

Hirota, Y., Mordoh, J., and Jacob, F. (1970) J. Mol. Biol. 53, 369—387.
Hirota, Y., Yamada, M., Nishimura, A., Oka, A., Sugimoto, K., Asada,
K., and Takanami, M. (1981) Prog. Nucleic Acid Res. Mal. Biol. 26,
33-47.

Hoyt, M. A., Knight, D. M., Das, A., Miller, H. I., and Echols, H. (1982)
Cell 31, 565-573.

Hwang, D. S., and Kaguni, J. M. (1988) J. Biol. Chem. 263, 10625-
10632.

Kaguni, J. M., LaVerne, L. S., and Ray, D. S. (1979) Proc. Natl. Acad.
Sci. USA 76, 6250-6254.

Kellenberger-Gujer, G., Podhajska, A. J ., and Caro, L. (1978) Mol. Gen.
Genet. 162, 9-16.

Kucherer, C., Lather, H., Kolling, R., Schauzu, M., and Messer, W.
(1986) Mol. Gen. Genet. 205, 115-121.

Kung, F. C., and Glaser, D. A. (1978) J. Bacteriol. 133, 755-762.

Lycett, G., Orr, E., and Pritchard, R. (1980) Mol. Gen. Genet. 178,
329-336.

Lather, H., Kolling, R., Kucherer, C., and Schauzu, M. (1985) EMBO J.
4, '555-560.

Maizels, N. (1973) Proc. Natl. Acad. Sci. USA 70, 3585-3589.

Ohmori, H., Kimura, M., Nagata, T., and Sakakibara, Y. (1984) Gene
28, 159-170.

Miki, T., Kimura, M., Hiraga, S., Nagata, T., and Yura, T. (1979) J.
Bacteriol. 140, 235-247.

Sakakibara, Y., and Mizukami, T. (1980) Mol. Gen. Genet. 17 8,
541-553.

35.
36.

37.

38.

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43.

90

Skarstad, K, Boye, E., and Steen, H. B. (1986) EMBO J. 5, 1711-1717.

Stuber, D., and Bujard, H. (1981) Proc. Natl. Acad. Sci. USA 78,
167-171.

Sompayrac, L., and Maaloe, O. (1973) Nature New Biol. 241, 133-135.

Stuitje, A., de Wind, N., van der Spek, J. C., Pars, T. H., and Meijer,
M. (1986) Nucleic Acids Res. 14, 2333-2344.

Tippe-Schindler, R., Zahn, G., and Messer, W. (197 9) Mol. Gen. Genet.
168, 185-195.

Wu, R. (1970) J. Mol. Biol. 51, 501-521.

Zyskind, J. W., Deen, L., and Smith, D. (1977) J. Bacteriol. 129,
1466-1475.

Kolling, R. and Lather, H. (1985) J. Bacteriol. 164, 310-315.
Lorimer, D. D. and Revzin, A. (1986) Nucl. Acids Res. 14, 2921-2938.

Chapter III
TRANSCRIPTIONAL REPRESSION OF THE maH GENE
OF Escherichia 9011 BY dnaA PROTEIN

91

ABSTRACT

The M (hth) gene of Whig c911 encodes a sigma factor which
confers upon RNA polymerase the ability to recognize the promoters for genes
responsive to the phenomenon termed the heat shock response. dnaA protein,
a sequence-specific DNA binding protein, is required for initiation of
chromosomal replication by binding to sites within the chromosomal origin.
dnaA protein also autoregulates its expression by binding to a site in the
dnaA promoter region. Two copies of the dnaA protein recognition sequence
are present within the mall promoter region. Using filter binding assays,
dnaA protein was observed to bind specifically to DNA fragments containing
the mall promoter region with greater affinity than its binding to the dnaA
promoter region. By contrast, reduced binding to a DNA fragment containing
the lacUV5 promoter was observed. DNase I footprint analysis indicated that
dnaA protein protected specific sites within the rum promoter region. The
binding of dnaA protein to the maH promoter region resulted in
transcriptional repression from two of the three promoters of the mnH gene
in vitro. Elevated levels of dnaA protein repressed transcription from these
two [pal-1 promoters in vivo. These results indicate that dnaA protein
regulates maH transcription to influence the expression of genes under maH
control.

92

INTRODUCTION

dnaA protein is required for initiation of DNA replication from the E. 9911
chromosomal origin, 21:10.. in vivo (26, 57, 32) and in vitro (17, 6). In this
process, dnaA protein recognizes and binds cooperatively to sites within the
mic region (11, 18). Upon binding, this protein appears to induce a localized
unwinding of am in an ATP-dependent fashion to create a structure required
for subsequent steps of replication (3, 46, 6). These and other results suggest
that dnaA protein functions at an early stage in the initiation process.

dnaA protein not only binds to MIC. but to other DNA fragments
including those containing the dnaA promoter, the 16 KDa promoter, and to
origin sequences of some plasmids (18). DNA fragments specifically bound by
dnaA protein share a nine base-pair consensus sequence, 'I'I‘AT(A/C)CA(A/C)A,
which is postulated to be recognized by dnaA protein. Other experiments
indicate that dnaA protein represses transcription of the dnaA and the 16
KDa genes (7, 2, 31, 36, 48, 54). This repressive effect was observed at levels
of dnaA protein required for specific binding to these promoter-containing
DNA fragments (54). The ability of dnaA protein to autoregulate its
expression and its role in initiation of E. can chromosomal replication may
contribute to regulating the frequency of initiation. The binding of dnaA
protein to plasmid origins has also been correlated with the involvement of
dnaA protein in plasmid DNA replication (25, 16, 43, 30, 24, 40, 47). These
observations suggest a biochemical function in. site-specific binding by dnaA

93

94
protein.

E. can exposed to high temperature is induced to express seventeen
specific proteins in a phenomenon termed the heat shock response (42). Other
treatments which elicit this response include ethanol, bacteriophage infection,
or UV irradiation. A sigma factor, a”, encoded by the [p.911 (11mm gene
confers upon RNA polymerase the ability to recognize and transcribe from the
promoters of these heat shock genes (33, 23, 14, 4).

Recent reports indicate that the level of 0'” is relatively low at 300 and
increases by a temperature shift to 42C (34, 49). The increase in 0'” appears
to occur by increased transcription and by stabilization of man mRNA (50,
15). 132.911 transcripts start at promoters designated here as mQH1P, 2P, 3P,
and 4P (15, 51). Promoters IP and 4P are recognized by tin-RNA polymerase
and account for 90% of the total rpoH mRNA at 30‘C (15). mH3P may be
recognized by a novel form of RNA polymerase. A fourth promoter, 2P,
appears to be strain dependent. A temperature increase results in an increase
in 31911 transcript levels with 2P and 3P increasing the most. Despite the
response of wall expression to increased temperature, its expression does not
appear to involve a” (4, 15).

Computer analysis revealed two presumptive dnaA protein recognition
sequences (dnaA boxes) in the promoter region of the 11911 gene. This
observation led us to consider whether dnaA protein might regulate
expression of the m gene.

In this study, we report that dnaA protein binds specifically to the MH
promoter region as determined by nitrocellulose filter binding assays and

95
DNase I protection experiments. dnaA protein repressed transcription from

two promoters of the anti gene in vivo and in vitro.

MATERIALS AND METHODS

B | . l | . i l 'i

E. 2011 W3110 and AB1157 131121, 3:144, 1:336, delmmmz, 13911,
tsx:33.snnEA4.zalK2.hiaGA.mLflLndzfi.thLmEfi.thmwerefi‘om
the E. 9911 Genetic Stock Center.

Plasmid pINGl (29) was from Dr. Dan S. Ray, UCLA. pDS596 contains
the dnaA gene inserted in the vector pINGl and under control of the m3
promoter (28). The plasmid pdnaA/dnaN contains the dnaA and adjacent
dnaK genes within the 0.94 and the 3.4 kb EcoRl fi'agnents inserted in the
vector pMOB45 (9). pFN82, from Dr. Frederick C. Neidhardt, University of
Michigan, consists of a 2.5 kb PstI fragment containing the N-terminal coding
region of the maH gene inserted at the single PstI site of pBR322 (41).
pBR322 and pUC 19 were from this laboratory. Plasmid DNAs were purified
from cleared lysates by centrifugation in ethidium bromide~CsCl gradients.
Individual restriction fragments separated by electrophoresis were purified by
gel electroelution with an ISCO 17 50 electrophoretic concentrator followed by
ethanol precipitation. DNA concentration was determined
spectrophotometrically, or by comparison of the purified DNA fragments to
different known amounts of electrophoretically separated restriction enzyme
digests visualized by ethidium bromide staining. The 203 bp EcoRI fragment
containing the lagUV5 promoter was a gift from D. Lorimer of this
department (35).

96

97

Enzymes
Restriction enzymes HindIII, EcoRl, ClaI, EcoRV, and Pqu, DNA poly

merase I (large fragment), and T4 polynucleotide kinase were purchased from
New England Biolabs; PstI was from BRL; HpaII and S1 nuclease from
Pharmacia; DNase I from Worthington. RNA polymerase holoenzyme was
purified from W3110 as described except that Biogel A5m (Biorad)
chromatography was replaced by chromatography on a TSK 3000SW (Altex)
high performance gel permeation column (10, 22). dnaA protein was purified
from an overproducing strain (28). Protein concentrations were determined by
the dye-binding method with bovine serum albumin as a standard (5).

B 1' I' I] 11' [ENE
Restriction enzyme digests or purified DNA fragments were either 5’

end-labeled with T4 polynucleotide kinase and [WP] ATP according to the
manufacturer’s instructions or 3’ end-labeled with DNA polymerase I (large
fragment) and [on‘P1dCTP as described (54).

DRE 1 . l'
Nitrocellulose filter binding assays were performed as described (54).
DNase I protection assays (19) (10 pl) contained the indicated amounts of
dnaA protein and end-labeled DNA fragments in buffer containing 40 mM
Tris-HCl pH 7.6, 100 mM NaCl, 10 mM MgCl.” 0.1 mM EDTA, 2 mM
dithiothreitol 100 ug/ml bovine serum albumin, and 10% glycerol. The

98

PvuII-ClaI fragment labeled at the 5’ end as described above was cleaved
with HpaII and used without further purification in these assays. Cleavage
with HpaII produces a 27 bp PvuII-HpaII fragment and a 156 bp HpaII-ClaI
fragment. For the other strand, the PvuII-ClaI fragment was 3’ end-labeled at
the ClaI site. Reactions were incubated at 37°C for 10 min. DNase I (4 ng)
was added and incubations were continued for 2 min. After addition of an
equal volume of stop bufi‘er containing 0.1% sodium dodecyl sulfate, 0.3 M
sodium acetate, 20 mM EDTA, and 100 ug/ml tRNA, the samples were
ethanol precipitated, resuspended in 5 ul of gel loading bufl‘er containing 80%
formamide, 10 mM NaOH, 1 mM EDTA and 0.025% bromophenol blue, boiled
for 2 min, then placed on ice. Electrophoresis was in an 8% sequencing gel.

Maxam-Gilbert sequencing reactions of the end-labeled DNA served as

markers for DNase I protection experiments (38).

B E | . |°
In vitro transcription assays were performed as described with the
following modifications (54). A prior incubation was performed at 37'C for 10

min in bufi'er containing 0.025 pmol of the indicated DNA fragment, 0.65
pmol of RNA polymerase, and the indicated amounts of dnaA protein but in
the absence of ribonucleotides. Heparin (100 ug/ml), 300 M each of CTP,
GTP, ATP, and 50 M [OPP] UTP were added to inhibit open complex
formation and to initiate transcription. Incubation was continued at 37C for
10 min. Reactions were stopped, and samples were ethanol precipitated,

resuspended, and electrophoresed in 8% polyacrylamide gels containing 7M

99
urea.

Gels were dried onto Whatman DE81 paper, and autoradiographed with
Kodak XAR-5 film either at room temperature or at -70‘C with Cronex
Quanta III intensifying screens. The autoradiograms were quantitated by
densitometry with a Hoefer gel scanner interfaced with an IBM personal

computer.

W

RNA was prepared (8) with the following modifications. E. 99.11 AB1157
containing either pINGl or pDS596 was grown in LB media (120 ml) (39) at
30‘C in a shaking water bath to a turbidity at 595 nm of 0.1. Arabinose was
then added to 0.75% (w/v) to induce synthesis of dnaA protein. Incubation
was continued at 3°C with shaking and portions were removed prior to or at
the indicated times after arabinose addition. Where indicated, cultures were
shifted to 42°C afier induced expression of dnaA protein for 2 hr at 300 and
portions were removed at the indicated times. Portions of the culture (3 ml)
from each time point were immediately centrifuged in a Fisher
microcentrifuge for 20 seconds at room temperature. Resuspended cells were
lysed as described (8). The lysate was phenol extracted three times, ether
extracted, and total cellular RNA was obtained by ethanol precipitation. RNA
concentration was determined spectrophotometrically (1 absorbance unit at
260nm equals 40 ug/ml).

100

amalgam

Quantitative S1 nuclease assays (20 ul) were performed in buffer
containing 40 mM PIPES pH 6.4, 1 mM EDTA, 0.4 M NaCl, 25 ug of yeast
tRNA (Sigma), and 80% formamide by hybridization at 45°C for 12 to 16 h
with 25 ug of the isolated RNA to 0.025 pmol of 5’ end-labeled EcoRV
restriction fragment containing the 131911 promoter region. At the end of
hybridization, 300 pl of cold S1 nuclease buffer containing 50 mM sodium
acetate pH 4.6, 0.25 M NaCl, 4.5 mM ZnSO4, 20 ug/ml heat-denatured
salmon sperm DNA and 50 units of S1 nuclease was added. Reactions were
incubated at 37‘C for 15 min. Ammonium acetate and EDTA were added to
0.25 M and 5 mM, respectively. The samples were precipitated by addition of
2.5 volumes of ethanol, and resuspended in 20 ul of gel loading buffer
described above. Electrophoresis was in an 8% polyacrylamide gel containing 7
M urea. Autoradiography, and its quantitation were as described above.

RESULTS

00:; our. 000: :O‘H.l= .0 ".6100! lz'JH‘t.‘ 005.00? 0‘ .00!
mmateuexian

Two presumptive dnaA protein recognition sequences (dnaA boxes) were
observed in the promoter region of the mall gene (Figure 1A,B). To examine
the binding aflinity of dnaA protein to the mH promoter region,
nitrocellulose filter binding assays were performed. Plasmid pFN82 contains
the promoter region and the N-terminal [pal-I coding sequence in a 2.5 kb
PstI fragment inserted at the PstI site of pBR322 (41). HpaII digestion of
pFN 82 results in fragments including those containing the rpm promoter
region (233 bp), and the pBR322 origin (527 bp). The latter DNA fragment
contains a dnaA box near the pBR322 origin. dnaA protein binds to this
fragment with an afiinity similar to its binding to the dnaA promoter region
(18; data not shown). Filter binding assays were performed with increasing
amounts of dnaA protein (Figure 2). A DNA fragment near the size expected
for the maH promoter-containing fragment (233 bp) was bound with higher
affinity by dnaA protein than its binding to the 527 bp pBR322 origin
fragment (Figure 2).

Based on DNA sequence information, HindIII digestion of the fragment
containing the Mn promoter (233 bp) is expected to produce two fragments
of 186 and 43 bp. A second HindIII site is located in the 622 bp HpaII
fragment of the vector. It is not bound by dnaA protein under these reaction

101

102

Figure 1. (A) Physical map of the mH promoter region. Pertinent restriction
enzyme sites: C, ClaI; H, Hind III; Hp, HpaII; Rv, EcoRV; Ps, PstI; Pv, Pqu.
The approximate positions of dnaA boxes (E ), maH promoters (no), and the
mail coding region (:3), are indicated. Restriction fragments used in the
experiments are also indicated.

(B) DNA sequence of the man promoter region (33, 56, 20) (coding
strand). The C-terminal coding region of flax, positions of transcriptional start
sites for {pol-I mRNA (15), dnaA protein recognition sequences (59), the
N-terminal coding region of man, and the restriction enzyme sites are as
indicated.

103

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Figure 2. Preferential binding of dnaA protein to a restriction fragment
containing the [22171 promoter region.

Filter binding assays were performed as described in Experimental
Procedures with the indicated amounts of dnaA protein and 100 ng of 3’ end-
labeled pFN82 digested with HpaII. M, 3’ end-labeled pBR322 DNA digested
with HpaII used as a size standard; TD, total digest of pFN82; B, bound
fragments eluted from the filter; F, fragments which flowed through the filter.
HindIII, DNA fragments retained on the filters by 100 ng of dnaA protein
were isolated, and digested with HindIII prior to electrophoresis.

105

dnaA protein 0 '2‘5 25 50 IOO
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106
conditions. DNA fragments bound with 100 ng of dnaA protein and retained
on a nitrocellulose filter were isolated and digested with HindIII restriction
enzyme (Figure 2). The 527 bp fragment containing the pBR322 origin and
lacking a Hinan site was not digested. Absence of the fragment near 233 bp
and production of subfragments of about 190- 195 bp and 40 bp by HindIII
confirm that the HpaII fragment bound with greatest aflinity by dnaA protein
contains the Mn promoter region.

The relative binding affinity of dnaA protein to fragments containing the
promoter regions of dnaA (945 bp), man (659 bp), and lacUV5 (203 bp) was
measured in filter binding assays (Figure 3). With equimolar amounts (in
fi‘agment) of each, higher affinity binding by dnaA protein to the [pal-I
promoter-containing fragment (659 bp) was observed compared to the dnaA
promoter-containing fragment (945 bp). The lacUV5 fragment (203 bp) was
poorly bound. The increased binding affinity to the 111011 promoter-containing
fragment may be due to the presence of two dnaA protein recognition
sequences in the mall promoter region compared to one such sequence in the

region of the dnaA promoters. Alternatively, nucleotides flanking the

recognition sequences may influence binding afiinity.

 

In order to locate the sites of binding by dnaA protein in the mofi
promoter region more precisely, DNase I protection assays (19) were
performed. Restriction fragments containing the mail promoter region were 5’
or 3’ end-labeled at the ClaI site and incubated with varying amounts of

107

Figure 3. Preferential binding of dnaA protein to restriction fragments
containing the dnaA promoter region, or the mat}. promoter region.

(A) Filter binding assays were performed as described in Experimental
Procedures with 0.025 pmol each of 5’ end-labeled DNA fragments containing
the promoter regions of dnaA, mall, and lacUV5. The amounts of dnaA
protein added are indicated. TD, total DNA used in the binding assay; B,
bound fragments eluted from the filter; F, fragments which flowed through
the filter.

(B) The fraction (in percent) of a DNA fragment retained and eluted from
filters is expressed as a ratio to the total amount of that fragment in both the
"bound" and "flow-through" lanes measured as described in Experimental

Procedures.

108

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109

Figure 4. dnaA protein binds to both dnaA boxes in the mH promoter
region.

DNase I protection assays were performed with the indicated amounts of
dnaA protein and (A) 3’ end-labeled PvuII-ClaI fragment, or (B) 5’ end-labeled
HpaII-ClaI fiagment as described in Experimental Procedures. The protected
regions, ( fl ), dnaA protein recognition sequences, and lanes containing the
products of Maxam-Gilbert sequencing reactions of the end-labeled DNA
fragments used in the DNaseI protection assays are also indicated.

110

.3 .5 .3 5

 

  

..5 3 5 3

TTATTCACA TGTGGATAA w

  

dnaA protein
(ng)

A.

111
dnaA protein. A limited digestion with DNase I was performed and the
denatured products were separated on a sequencing gel (Figure 4A,B). Four
regions of protection were observed on either strand in the vicinity of the two
nine base pair dnaA boxes. This pattern of protection was observed at levels
of dnaA protein sufiicient for specific retention of 19911 promoter-containing
fragments on nitrocellulose filters.

 

Transcription of the mH gene apparently involves as many as four

promoters (15, 51). maHlP and 4P are recognized by (SN-RNA polymerase. A
third promoter, 3P, is apparently recognized by a novel form of RNA
polymerase which may be distinct from o”-, or o”-RNA polymerase. Detection
of the fourth promoter, 2P, by S1 mapping was dependent on the E. £011
strain from which the RNA was isolated (15).

Difi‘erent restriction fragments were used in run-off transcription assays
to define the start sites and strandedness of the transcripts, and to determine
the influence of dnaA protein on [p.911 transcription. Run-ofi‘ transcription
assays from the mat! promoters were performed with various preparations of
(I'm-RNA polymerase purified as described (15, 22). Most preparations resulted
in transcription from 1? and 4P (data not shown). This is consistent with
results of others that a”- RNA polymerase mediates transcription from these
promoters. One preparation resulted additionally in transcription from 3P

presumably due to the presence of a novel factor sigma or a positive

112
regulatory protein. Due to the proximity of the dnaA protein recognition
sequences to 3P, this preparation of RNA polymerase was used in the
following experiments.

The DNA templates used included the PvuII-ClaI, PstI-ClaI, and EcoRV
fragments containing the mH promoter region (Figure 1). Transcription of
the 659 bp PvuII-ClaI fragment resulted in three transcripts of about 220, 75,
and 65 nucleotides relative to single-stranded DNA marker fragments
electrophoresed in a separate lane (Figure 5). The PstI-ClaI fragment which
extends 1.2 kb upstream fi'om the PvuII site was used as a template. Three
transcripts were observed with this template and were of similar sizes as
those with the PvuII-ClaI fragment. Compared to the PvuII-Clal fragment,
the 342 bp EcoRV fragment is truncated by four nucleotides on the template
strand near the N-terminal mafi coding region (Figure 1B) and is about 320
bp shorter at the other end. Three transcripts observed with the EcoRV
fragment were each slightly shorter than those observed with the PvuII-ClaI
fragment. ClaI restriction within the mH promoter region of the template
DNA strand produces a 5’ end 16 nucleotides from the first codon. Taking
this distance into account, these experiments indicate transcriptional start
sites at approximately 235, 90, and 80 nucleotides upstream from the coding
sequence. Whereas these transcripts have not been mapped relative to the
products of sequencing reactions of the template fragment, these results are
consistent with results of others (15, 51) in identification of promoters 1P, 3P,
and 4Pa (referred to henceforth as 4P) respectively (Figure 1). Transcripts

from 2P or 4Pb were not observed in this or other experiments (Figure 6,

113

Figure 5. Run-off transcription assays with DNA fragments containing the
mH promoters.

Assays were performed as described in Experimental Procedures with the
indicated DNA fragments as templates. The positions of transcripts from the
mold, promoters are indicated. M, 3’ end-labeled pBR322 DNA digested with
HpaII was included as a size standard.

114

H
2
U
l

H
o—
(n
0.

EcoRV - EcoRV

 

115

Figure 6. dnaA protein inhibits transcription from mH3P and mH4P.

(A) Run-off transcription assays were performed as described in
Experimental Procedures with the 203 bp 13le fragment containing the
lacUV5 promoter or the 1891 bp PstI-ClaI fragment containing the maH
promoter region. The amounts of dnaA protein added are indicated. The size
standard was as in Figure 5.

(B) The relative amounts of each transcript determined as described in
Experimental Procedures are expressed as a ratio compared to the amount

produced with no dnaA protein added.

116

as zmcbmn. <95

00. m» Om 0N
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8 9
o' 0'
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'5
O

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<2

 

 

 

 

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117
data not shown). Such experiments should have resolved 4Pa transcripts (82
or 83 nucleotides) from 4Pb transcripts (78 nucleotides). Radioactive products
near the top of the autoradiogram (and in Figure 6A) are presumably due to
end-to-end transcription of the restriction fragment.

Run-ofl‘ transcription assays were performed with the PstI-ClaI fragment
to determine the influence of dnaA protein on {95111 transcription (Figure
6A,B). Transcription from mQH4P was markedly inhibited with comparatively
less inhibition from 3P with increasing amounts of purified dnaA protein
added. At these levels of dnaA protein, specific binding of dnaA protein to the
mgH promoter region was observed (Figure 2,3). Similar results of
transcriptional inhibition of maH3P and 4P by dnaA protein were obtained
with the HpaII or EcoRV DNA fragments containing the anti promoter
region (data not shown). No inhibition by dnaA protein was observed on
transcription from mngP.

Run-ofl‘ transcription assays were also performed with a 203 bp
restriction fragment containing the lagUV5 promoter. By sequence analysis,
this DNA fragment lacks sequences similar to the consensus dnaA protein
recognition sequence, and is poorly bound by dnaA protein (Figure 4). The
observed transcript of about 67 nucleotides is consistent with transcription
from the lacUV5 promoter in the absence of cyclic AMP binding protein
(Figure 6A) (37). Addition of increasing amounts of dnaA protein to these
assays marginally afi’ected the synthesis of this transcript (Figure 6B). The
product of about 240 nucleotides is presumably due to end-to-end
transcription of the restriction fi'agment. Its slightly greater apparent size

118
appears to be due to its anomalous electrophoretic migration in this
experiment compared to other experiments of run-off transcription with this
fragment (54; data not shown). Other high molecular weight products are
unexplained but have been observed by others in transcription of this
fragment (35).

ii |°'l']'| HI 'I' . .
The plasmid pDS596 contains the dnaA gene under inducible expression

from the m3 promoter (28). In the uninduced state, transcription from the
m3 promoter is repressed by the M gene product encoded by the vector
pINGl. Based on the above in vitro results, increased levels of dnaA protein
by induced expression of the cloned dnaA gene were expected to inhibit {pal-I
transcription.

E. 9911'. AB1157 containing either pDS596 or the vector pINGl was grown
in LB media at 30’C. Expression from the amB, promoter was induced by
addition of arabinose. Total cellular RNA was isolated from portions of each
culture removed prior to and at various times after addition of arabinose.
RNA was hybridized to an excess of 5’ end-labeled EcoRV restriction fragment
containing the mall promoter region, treated with S1 nuclease,
electrophoresed, and autoradiographed (Figure 7A). The relative amounts of
each transcript from the 1191-1 promoters were determined (Figure 7B).

S1 mapping experiments with RNA from E. nah AB1157 containing the
vector pINGl indicated a relatively constant level of transcript from mHlP

prior to and at various times alter addition of arabinose. Its addition

119

Figure 7. Inhibition of [pol-MP transcription by overproduction of dnaA
protein in vivo.

(A) E. 99.11 A3115? harboring either the vector pINGl, or pDS596
containing the dnaA gene were grown in LB media at 30°C to a turbidity at
595 nm of 0.1. Arabinose was then added to 0.7 5% (w/v) to induce synthesis
of dnaA protein. RNA was isolated from portions of each culture removed
prior to or at the indicated times after arabinose addition and used in $1
mapping experiments as described in Experimental Procedure. The molecular
weight markers were 3’ end-labeled pUC 19 DNA digested with EcoRI and
HpaII.

(B) mHlP and 4P transcripts were quantitated as described in
Experimental Procedures. The level of each respective transcript is expressed
as a ratio compared to that contained in RNA from AB1157 harboring the
vector pINGI and removed just prior to the time of arabinose addition. Open
symbols (A’o), moi-I from AB1157 containing pINGl; closed symbols 0.0). M
mRNA from AB1157 containing pDS596.

120

om.

Om.

2E 2.:

Om

 

 

 

 

 

on.

m. dMIoEI l5

avzoe m
_ q W lo:

0
W IN?—
1. EIGQEIR‘iloa.
IN¢N
0305.... Ilu'llll':l'll'llll”v.n
lam

 

 

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121
appeared to result in a transient increase in 4P transcript at 10 min before
returning to the original level. The reason for this transient increase is not
understood. Transcripts initiating from 3P were barely detectable compared to
IP and 4P. Whereas transcripts from 2P were not detected, the relative levels
of other mH transcripts prior to arabinose addition are in agreement with
observations of Erickson et a1. (15), and Tobe et a1. (51). The influence of
arabinose addition on 3P transcription was not determined due to its low
abundance prior to arabinose addition. The Sl-resistant material migrating
near the bottom of the gel and near 100- 130 nucleotides appeared to have
arisen by hybrid formation between the radioactive probe and yeast tRNA
and/or the denatured salmon sperm DNA included in the reactions (data not
shown).

81 mapping experiments were performed in parallel with RNA isolated
from AB1157 harboring pDS596 prior to and at various times after induction
(Figure 7A). As above, a transient increase in the level of 4P transcript was
observed 10 min after addition of arabinose (Figure 7 B). Almost no mH4P
transcript was detected by thirty minutes of induced expression of dnaA
protein. This interval corresponds to the time after induction when the
replication activity of dnaA protein was detectable in extracts from similar
cultures (Hwang and Kaguni, unpublished results). Levels of 4P transcript
were further reduced at later time points. By contrast, the relative level of 1P
transcript was marginally altered by elevated levels of dnaA protein. As
above, the efi‘ect of elevated levels of dnaA protein on transcription from
mH3P was not determined due to its low abundance prior to arabinose

122
addition.

Reports indicate that the levels of moH transcripts increase with an
increase in temperature (50, 15). Transcripts from 2P and 3P were observed
to increase the most (15). This increased transcription contributes, in part, to
elevated expression of heat shock genes upon a temperature upshift. To
determine whether elevated levels of dnaA protein would inhibit this increase
in transcript levels, AB1157 containing either pDS596 or pINGl were grown
at 300 in LB media, arabinose was added, and induced expression of dnaA
protein proceeded for 2 hr at 30°C. The culture was then shifted to 42C. RNA
was isolated from portions of each culture prior to and at various times after
the temperature shift.

SI mapping experiments with RNA from AB1157 containing the vector
pINGl showed a two- to three-fold increase with the temperature shift in the
level of mHIP transcripts (Figure 8A,B). Consistent with previous
observations (15), 3P transcripts were most dramatically elevated by the
temperature shift. Due to the low levels of 3P transcript detected prior to the
temperature shift, it was not possible to quantitate accurately the relative
increase upon temperature upshif’t. In addition, an increase greater than
15-fold in the level of 4P transcript was observed 15 to 20 min after the
temperature shift. Transcriptional start sites mapped by 81 analysis on
sequencing gels for mngP, 3P, and 4Pa were in agreement with Erickson et
a1. (15; data not shown). Transcripts from 4Pb were not detected. As in
Figure 7A, the Sl-resistant material near the bottom of the gel and near
100-130 nucleotides was attributed to annealing between the probe and yeast

 

123

Figure 8. Inhibition of mH3P and mH4P transcription by dnaA protein
after a temperature shift.

(A) E. mli AB1157 harboring either the vector pING1, or pDS596
containing the dnaA gene were grown in LB media at 30‘C to a turbidity at
595 nm of 0.1. Arabinose was added to 0.75% (w/v), the cultures were
incubated at 30°C for two hours then shifted to 42°C. RNA was isolated from
portions of each culture prior to or at the times indicated after the
temperature shift. SI nuclease mapping experiments were performed as
described in Experimental Procedures. The size standard was as in Figure 7.

(B) mHlP and 4P transcripts were quantitated as described in
Experimental Procedures and normalized to the level of each respective
transcript contained in RNA from AB1157 harboring pINGl at 300. Open
symbols 01,0), mH mRNA from AB1157 containing pINGl; closed symbols (A, O
), mQH mRNA from AB1157 containing pDS596.

3.5 we:

 

124

 

 

 

 

 

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E.
\ _
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xx In
a nice
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125
tRNA and/or the denatured salmon sperm DNA included in the reactions
(data not shown). Transcripts from 2P were not observed.

81 mapping experiments with RNA isolated from ABll57 containing
pDS596 indicated that mHlP transcripts increased to between two— to
three-fold (Figure 8A,B). This result was similar to levels observed with RNA
from ABll57 containing the vector. By contrast, the large increase in 3P and
4P transcripts was not observed after the temperature shift. Based on other
experiments (28), this strain presumably contains elevated levels of dnaA
protein which inhibits transcription from 3P upon the temperature shift. For
reasons described above, the levels of 3P transcripts were not quantitated.
While the amount of 4P transcripts increased to three- fold with time after
the temperature shift, levels of this mRNA were far less than those observed
with AB1157 containing the vector pINGI at similar times. These results
indicate that dnaA protein preferentially inhibits transcription from an3P

and 4P in vivo.

DISCUSSION

Upon a shift to high temperature, E. 9911 expresses a set of about
seventeen proteins in a phenomenon termed the heat shock response. This
response appears to have been conserved in all organisms suggesting its
essential role in cell physiology.

Due to the presence of two presumptive dnaA protein recognition
sequences in the 193111 promoter region, we were interested in determining
whether dnaA protein would specifically bind to these sites to influence mH
expression. The results shown here indicate that dnaA protein specifically
bound to the mgH promoter region to repress transcription from promoters 3P
and 4P. This inhibition may involve occlusion of RNA polymerase by dnaA
protein. That transcription from 1P was not influenced suggests that dnaA
protein bound at a down stream site does not interfere markedly with the
progress of a transcribing RNA polymerase. This result is consistent with the
observation that lac repressor bound at a distal site only transiently blocks a
transcriptional elongation complex (44, 27).

Although dnaA protein can be isolated as a monomer, its form as a
transcriptional repressor remains to be determined. In studies of dnaA protein
binding to the dnaA promoter region (18, 7), a complex DNase I footprint was
observed which extended 40 to 50 bp on either side of the dnaA protein
recognition sequence. This result appears to have been due to addition of
excess amounts of dnaA protein. At levels of dnaA protein (2 pmol of protein

126

127
with 0.025 pmol of fragment) suflicient to specifically inhibit 32ng (this study)
and dnaA transcription (54; unpublished results), protection from DNase I
was limited to a 30 to 40 bp region encompassing the dnaA protein
recognition sequence. In comparison, lac repressor, a tetramer of identical
subunits, protects a 25 bp region when bound to its operator ( 19).

The role of the heat shock response is not well understood. Proposed
models relate the heat shock response to thermotolerance, protein
degradation, or cell division. In E. c911, thermotolerance upon temperature
upshift is dependent on a brief incubation at an elevated but nonlethal
temperature (55). In this previous study, exposure of cells to this intermediate
temperature was presumed necessary for expression of heat shock proteins.
However, induction of the {pol-I gene product under control of the inducible
tag promoter at low temperature did not confer thermotolerance at high
temperature (53). Thermotolerance did not appear to depend on
mil-dependent expression of heat shock proteins.

The production of abnormal proteins in E. can by incorporation of
puromycin, the arginine analog, canavanine, or by induced synthesis of a
foreign protein results in elevated levels of heat shock proteins (21). Other
conditions which induce the heat shock response may also generate abnormal
polypeptides (1). Since Lon, an ATP-dependent protease (13, 12), is a heat
shock protein, the heat shock response may function in protein degradation.

The role of the heat shock response in cell division is based on studies of
null mutants. Whereas mH nonsense (and some missense) mutants are

viable up to 300 in nonsuppressing strains due to the production of trace

128
amounts of a”, an mflzzkan mutant is viable only at or below 200 (51).
6'” appears to be essential at higher growth temperatures. When such
mutants are shifted to 420, cell division stops with an increase in mass to
form long filaments (52).

Other treatments which result in expression of heat shock proteins
include ethanol, UV irradiation, or bacteriophage infection. That dnaA protein
negatively regulates mfl expression suggests that these treatments may
inactivate dnaA protein as a repressor or, in the case of bacteriophage
infection, that a site(s) on the replicating viral DNA or a viral gene product
acts as a competitor for binding of dnaA protein.

In E. 9911 as in many organisms, DNA replication and cell growth are
tightly coordinated events. dnaA protein functions at an early step in
initiation of DNA replication from the chromosomal origin, mg (57 , 32, 6).
That expression of mail is influenced by this initiator protein for DNA
replication and that moH mutants render the cell incapable of proper cell
division (52) suggest a regulatory mechanism whereby dnaA protein may
coordinate initiation of DNA replication to the expression of genes under
control of m which are required for cell division.

With regard to this, an allele of dnaK, a heat shock gene, has been
isolated which is conditionally defective in initiation of DNA replication (45).
We have recently determined that dnaK protein stimulates the replication
activity of a mutant form of dnaA protein (Hwang and Kaguni, unpublished
results). dnaK protein also appears to influence the activity of wild type dnaA
protein in replication (Carr and Kaguni, unpublished results). The ability of

129
dnaA protein to regulate mQH expression may influence the level of dnaK
protein which in turn influences the activity of dnaA protein in initiation.

PS9?"

10.
11.

13.

14.

15.

16.

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Chapter IV
A NOVEL SIGMA FACTOR INVOLVED IN EXPRESSION

OFTHEmHGENEOFEscherichiacnli

134

ABSTRACT

The E. 99.11 131911 gene encoding 0” involved in the heat shock response is
transcribed by as many as four promoters. We have isolated a novel sigma
factor of about 24 KDa from a preparation of RNA polymerase by
electroelution from SDS-polyacrylamide gels. The renatured protein conferred
upon core RNA polymerase the ability to transcribe preferentially from an
man promoter which is regulated by dnaA protein. Whereas the stringent
response which induces expression of heat shock proteins resulted in a
decrease in transcript levels from this promoter, other mH transcripts were
not elevated. This result suggests that induction of heat shock proteins by the
stringent response is not mediated by increased transcription of the mail

gene.

135

INTRODUCTION

The heat shock response in E. coli induced by such treatments as a
sudden temperature upshift, bacteriophage infection, or UV irradiation results
in preferential synthesis of at least 17 heat shock proteins (reviewed in 28).
The 11911 gene product, 6", plays a central role in this response by conferring
upon RNA polymerase the ability to initiate transcription from promoters of
heat shock genes (8, 15, 22). Upon temperature upshift, the increased
expression of heat shock proteins is attributable to an increase in a” due to
increased expression and stability (10, 12, 32, 33). A reprogramming of RNA
polymerase ensues.

Recent experiments have examined transcription of the 294111 gene (10,
12, 33). Four promoters designated mHlP, 2P, SP, and 4P have been
mapped at about 220, 130, 90, and 80 bp, respectively, upstream from the
coding sequence (10, 12). Promoters 1P and 4P are recognized by o”-RNA
polymerase (Eon) (3, 10, 12). A third promoter, moH2P appears to be
strain-specific. In vitro transcription from the fourth promoter, moHSP, does
not involve Eo’o or E0” (3, 10). These results suggest that a novel sigma
factor or a positive regulatory protein may be required for transcription from
moH3P. In related experiments examining regulation of the mgH gene, we
identified a preparation of RNA polymerase able to initiate transcription from
mHlP, 3P, and 4P (35). From this, we have isolated a protein of 24 KDa
which confers mQH3P specific transcription to core RNA polymerase. We also

136

137
report on transcriptional regulation of the mall gene during the stringent

response.

MATERIALS AND METHODS

B | .1 | . 1] 'i
E.snliW3110andAB1157thn:Lara;14.lenE§.delizntanmA162.lacYL

M.M.anZ.hiflfi$mfim.m:imtfl.mEfi.thklwerefi‘om
the E. £0.11 Genetic Stock Center. Plasmid pFN82 was from Dr. Frederick C.

Neidhardt (University of Michigan). To construct this plasmid, a 2.5 kb PstI
fragment containing the mall promoter region and adjacent N-terminal coding
region was inserted at the single PstI site of pBR322 (27). Plasmid pUC19
was from this laboratory. After restriction and gel electrophoresis, DNA
fragments were purified by electroelution with an ISCO 17 50 concentrator
followed by ethanol precipitation. The DNA concentration of a purified
fragment was determined by comparison to difl'erent amounts of
electrophoretically separated restriction enzyme digests stained with ethidium

bromide.

Enzymes
Restriction enzymes EcoRI, EcoRV, ClaI, and T4 polynucleotide kinase

were purchased from New England Biolabs; PstI was from BRL; HpaII and
SI nuclease were from Pharmacia. Samples of Ed"0 or core RNA polymerase
designated halo B and care B, respectively, lacking this novel sigma factor

were purified from W3110 as described (6, 14). This procedure, modified by
chromatography on a TSK 3000SW (Altex) high performance gel permeation

138

139
column instead of Biogel A5m (Bio-Rad), may have resulted in the presence of
this novel sigma factor in the resultant core and holoenzyme fractions cited as
core A and halo A, respectively. a” was obtained by separation of 07° from
E0” on a Bio-Rex 70 (Bio-Rad) column (6). Protein determination was by the

method of Bradford with bovine serum albumin as a standard (4).

Ell' E I'fi SDS] 1.1]
The preparation of core RNA polymerase containing the novel sigma

factor (1 mg of core A) was electrophoresed on a 10% sodium dodecyl sulfate
(SDS)—polyacrylamide vertical slab gel (15 cm x 15 cm x 1.5 mm) essentially
as described (17). The gel was cut into 10 fiactions to separate proteins by
size. Proteins were electroeluted using an ISCO 17 50 concentrator. The eluted
samples were divided into two equal parts, and precipitated by four volumes
of acetone (HPLC grade). One part of each fraction was resuspended in 10 ul
of 67 mM Tris-HCl pH 7.0, 0.001% bromophenol blue, 10% glycerol (v/v), 5%
mercaptaethanol (v/v), and 2% SDS, boiled for two minutes, separated on a
6%-20% SDS-polyacrylamide gradient gel, stained with Coomassie brilliant
blue, then silver stained (36) to visualize proteins. The other part was
renatured in 500 1.11 as described (17), and assayed for moH3P stimulatory
activity by addition to run-ofi' transcription assays containing core RNA
polymerase (core B). The recovery of protein judged by Coomassie blue
staining, and activity of this novel sigma factor relative to the sample loaded
on the SDS-polyacrylamide gel was about 10-20% and 8%, respectively.

140

W

Run-off transcription assays were performed with 0.025 pmol of the
PstI-Clal fragment and 0.65 pmol of RNA polymerase, unless indicated, in 10
ul of transcription bufl'er (34) at 37°C for 10 minutes in the absence of
ribonucleotides. Heparin (100 ug/ml), 300 M each of ATP, GTP, CTP, and 50
M of [OFF] UTP (2.5 uCi) (New England Nuclear) were added to a final
volume of 11 pl to inhibit further open complex formation and to initiate
transcription. Incubation was continued at 37°C for 10 minutes. Reactions
were then stopped, and samples were ethanol precipitated, resuspended, and
electrophoresed in an 8% polyacrylamide gel containing 7 M urea. Open
complex formation was in 5 1.11 when [”S] UTPaS (New England Nuclear) was
used as the radioactive nucleotide (1,37). Heparin, ATP, GTP, and CTP at the
above concentrations, and 0.5 uM (2.5 uCi) of [”S] UTPocS were added to a
final volume of 5.5 ul. After incubation at 37°C for 5 minutes, UTP was added
to 300 uM and the reactions (6 111) were further incubated for another 10
minutes to complete one cycle of transcription. An equal volume of buffer
containing 80% formamide, 10 mM NaOH, 1 mM EDTA, and 0.025%
bromophenol blue was added to stop the reactions. Samples were incubated at
100‘C for 2 min and electrophoresed as described above. Dried gels were
autoradiographed with Kodak KAR-5 film.

I l I' E I] | . |
ABll57 was grown to a turbidity at 595 nm of 0.3 in M9 minimal media
(26) supplemented with 0.4% glucose and 18 L-amino acids (without valine

141
and isoleucine) each at 100 ug/ml (21). The culture was then split into two
equal parts. RNA was isolated from portions of each culture (10 ml) removed
before and at various times after the addition of valine (500 ug/ml), or valine
and isoleucine (each at 500 ug/ml) (5). Valine addition results in isoleucine
limitation. The efficacy of the stringent response by valine addition was
confirmed by the reduction of M mRNA of about 20 fold which encodes
ribosomal protein L34 (data not shown). As a control, simultaneous addition
of isoleucine and valine did not markedly change rpmH transcript levels
measured by S1 nuclease protection assays.

D | l' E . . I] . i | . |
Quantitative Sl nuclease protection assays were performed as described

(35) by hybridization at 450 for 12-16 hr in 20 ul of bufi‘er containing 40 mM
PIPES pH 6.4, 1 mM EDTA, 0.4 M NaCl, and 80% formamide with 50 ug of
the isolated RNA to 0.025 pmol of 5’ end-labeled EcoRV restriction fragment
containing the M promoter region. The restriction fragment was confirmed
to be present in excess (data not shown). After hybridization, 300 pl of cold
81 bufl‘er containing 50 mM sodium acetate pH 4.6, 0.25 M NaCl, 4.5 mM
ZnSO4, 20 ug/ml of denatured salmon sperm DNA and 50 units of SI
nuclease were added. Reactions were incubated at 37'C for 15 minutes.
Ammonium acetate and EDTA were added to 0.25 M and 5 mM, respectively.
The samples were then ethanol precipitated, resuspended, electrophoresed,
and autoradiographed as described above.

RESULTS

11 I'fi I. E t} I . H3 HBEI .I.
The mH promoters are contained in a 1.89 kb PstI-ClaI restriction

fragment (10, 12) (Figure 1). While coding sequences for flax and part of M
are present, the promoter for this operon is not (13). Run-ofl‘ transcription
assays with this fragment were performed with various Ea” preparations
purified as described (6, 14). Such preparations have been shown to contain
a“, and dnaK protein among others (23, 31). Most preparations examined
resulted in RNAs of 210 and 67 nucleotides and correspond to transcription
from 1P and 4P. These promoters are recognized by Ed” (10). The results
from such a preparation of E0” (halo B) are in Figure 2. RNA polymerase
purified by a modification of the above procedure (Materials and Methods)
was chromatographed to separate the core enzyme from E0” (14). The
holoenzyme fraction (halo A) was able to transcribe not only from IP and 4P
but also from 3P to farm a 72 nucleotide RNA (Figure 2). Transcription fi'om
3P with this and the core fraction (core A) were presumably due to the
presence of a contaminating factor (see below). Due to residual amounts of
a” in the core fraction, low levels of 1P transcript also were observed. The
strandedness of these transcripts was confirmed by use of different DNA
fragments (35, data not shown). Transcripts near the top of the gel
presumably resulted from nonspecific transcription. The 190 nucleotide

transcript is unexplained and has not been detected in similar

142

143

Figure 1. Physical map of the anti promoter region (13,22,38).
Approximate positions of relevant restriction sites, @911 promoters b5),

coding sequences, polarity (lb) of the @911, flax, and ME genes, and
restriction fragments are indicated.

144

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an .mm. HEUAGQT

 

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in”. avian?

AlnHu £9.38

AAIDQNESL

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H20 >m8m >moom :wn.

145

Figure 2. Run-off transcription assays with preparations of RNA polymerase.

Assays were performed as described in Materials and Methods with the
indicated preparations of RNA polymerase. Transcripts from the @5211
promoters and sizes of denatured restriction fragments of pUC19 digested
with HpaII and EcoRI are indicated.

146

 

 

147

experiments(35).

 

Preliminary experiments indicated that this transcription factor was
relatively heat-stable (data not shown, see below). This property was
utilized to address its mechanism of action. If this factor functions as a sigma
factor, its addition to a preparation of core enzyme is expected to confer
specific transcription from moH3P. A sample of core RNA polymerase
containing this transcription factor (core A), when heated at 65°C for 5 min.,
was unable to transcribe from moHlP, 3P, or 4P (Figure 3). An unrelated
preparation of core RNA polymerase (core B) was obtained which cannot
initiate transcription from 3P. This preparation contained low levels of a” as
indicated by the relative level of transcription from moi-11R Upon addition of
the heat treated sample to this core fraction, transcription from 3P was
observed suggesting that this stimulatory factor acts as a novel sigma factor
(see below also). This presumed sigma factor retained about 70% of its
transcriptional activity after heat treatment.

I] HBEI .I. ill . I. EZIKD
To determine the size of this sigma factor, the core enzyme preparation

containing this activity (core A) was electrophoresed in an SDS-polyacrylamide
gel. Proteins were electroeluted from gel slices and renatured as described

(17 ). Renatured fractions were assayed for 3P transcriptional activity by
addition to core enzyme (core B) lacking this activity (Figure 4A). Most of the

 

148

Figure 3. The sigma factor which confers moH3P recognition is heat stable.

A preparation of core RNA polymerase (core A) containing the mHBP
stimulatory factor was not heated, or heated at 65°C for 5 min. in 10 pl of
transcription buffer in the absence of DNA. The untreated or treated samples
were assayed in run-off transcription (11 pl reaction volume) in the presence
or absence of a preparation of core enzyme (core B) lacking this stimulatory
activity. The reaction with both core B and heat treated core A were with
0.65 pmol of each.

149

OH
H

_ _rp0

< 98 8.60; + m 98 ..
m 9.8 1

SE m\oom® < 9.8 I

< 28 r

"rpoH/p
4 If 3
' 45

.. . ... F . . wax w

. . u u. . ......l §.unr.vn~.ur.fi:m
.C.~....,. 1...;

 

150

Figure 4. Isolation of a novel sigma factor by electroelution from an
SDS-polyacrylamide gel.

The core enzyme preparation (core A) containing the stimulatory factor
was electrophoresed in a 10% SDS-polyacrylamide slab gel. The gel was
fiactionated into 10 slices. Proteins were recovered by electroelution as
described in Materials and Methods.

(A) Proteins from each fraction indicated were renatured and assayed for
mH3P sigma factor activity in run-ofl‘ transcription assays with ["SlUTP by
addition of 1 pl to a preparation of core enzyme (core B) lacking this sigma
factor. Control assays were with RNA polymerase preparations containing this
activity (halo A, core A). Transcripts from mHlP, 3?, and 4P are indicated.

(B) Proteins recovered from the indicated fractions were electrophoresed on
an SDS-polyacrylamide gel as described in Materials and Methods and silver-
stained. Positions of the 24 KDa sigma factor, and molecular weight markers
bovine serum albumin (67 KDa), and trypsinogen (24 KDa) are indicated. For
comparison, 20 pg of the core enzyme preparation (core A) from which this
sigma factor was isolated was also electrophoresed on this gel.

   

588 _
b ODXVN 1 . m..-“ t OOXVN
ll . . 1.9.5 _ ates

 

__ _ _l._.
co=uc¢n QmmNmm¢MN_

. _ __
m cozocim mm
J . . J Jl
a m 8 90
V V 8V

mmuoo I+++++++++++I

152
3P transcriptional activity was in fraction 3. A minor amount was in fraction
2. A silver-stained polyacrylamide gel of proteins recovered from these
fractions revealed two bands near 24 KDa (Figure 4B). As fraction 4 was
enriched in the larger polypeptide contained in fraction 3 yet lacked the
activity of this novel sigma factor, it is unlikely that this polypeptide
represents this novel sigma factor. The presence of the smaller polypeptide in
fractions 2 and 3 correlates well with activity levels in these fractions. This
protein migrated slightly faster than trypsinogen of 24 KDa. Related
experiments in which the gel was cut into narrower slices to separate these
two polypeptides of similar sizes confirmed that the smaller polypeptide is
this novel sigma factor (data not shown). Assuming that polypeptides are
equally silver-stained, this sigma factor is a trace contaminant relative to

other contaminating polypeptides in this core enzyme preparation.

D 2| KB I . I . I} I
Sigma factors of RNA polymerase are physically associated with the core
enzyme to confer promoter recognition. Whereas other contaminating
polypeptides are present in the core preparation (Figure 4B), this novel sigma
factor in preparations of core and holoenzyme (Figure 2) suggests its physical
association. Experiments were performed to examine whether the renatured
24 KDa protein would compete with a" in interacting with the core enzyme.
Increasing levels of core enzyme (core B) lacldng this novel activity were
added to run-ofi‘ transcription assays containing a constant level of the
renatured polypeptide (Figure 5). Transcripts from 3P increased then

153

Figure 5. Competition between the 24 KDa protein and a” for core enzyme.

A constant amount (estimated to be less than 1 ng from the Coomassie
blue stained gel) of the renatured 24 KDa protein and increasing amounts of
core B, as indicated, were mixed in the absence or the presence of 50 ng (0.7
pmol) of 07° in run-ofi' transcription assays. Assays were performed as
described in Materials and Methods with [”S-thiollUTP. Transcripts from
moHlP, 3P, and 4P are indicated.

154

core B (ng)

 

155
remained relatively constant with increasing amounts of core enzyme
indicating that the 24 KDa protein was limiting at higher levels. The increase
in transcription from moHlP was presumably due to increasing levels of 07°
by addition of this core enzyme preparation.

In the presence of constant levels of 67° and the renatured 24 KDa
polypeptide, increasing amounts of core enzyme resulted in increased
transcription from mHlP and 4P. At low levels of care RNA polymerase,
reduced transcription from mH3P was observed in the presence of excess 6"
relative to assays lacking it. This result indicates that a" competes with the
24 KDa protein in interacting with the core enzyme and that this 24 KDa

protein acts as a sigma factor.

5' 2|. I'll'll I. II I. I.
The stringent response in E. 9911 can be induced by amino acid starvation

(reviewed in 7 ). A protein, stringent starvation protein, is predominantly
synthesized (29). This protein, stably associated with (I'm-RNA polymerase (20)
and formerly thought to be 22.5 KDa, is calculated to be 24.3 KDa from the
DNA sequence of the gene (30). It migrates at the F24.5 position on
two-dimensional polyacrylamide gel electrophoresis (30). Amino acid starvation
also induces synthesis of heat shock proteins (16). We considered that the
stringent starvation protein might be identical to this novel sigma factor. If
so, its elevated levels may result in increased transcription from mH3P
under the condition of stringent response. Experiments were performed to

determine whether 6" was identical to stringent starvation protein.

156

A culture was grown in a synthetic medium and divided. The stringent
response was induced by addition of valine to one part of the culture to
inhibit isoleucine biosynthesis. As a control, both valine and isoleucine were
added to the other half. Transcriptional regulation of the mall gene was
measured by $1 nuclease protection assays with RNA isolated from portions
of each culture removed at the indicated times (Figure 6). null transcript
levels were only slightly influenced by valine and isoleucine addition in the
control culture. By comparison, induction of the stringent response resulted in
a striking reduction in mHSP transcripts whereas IF and 4P transcript
levels were marginally affected. These results suggest that stringent
starvation protein and 0'" are not identical.

157

Figure 6. Repression of mgH3P under amino acid starvation.

Cells were grown at 37°C in glucose minimal media supplemented with
18 amino acids (without valine and isoleucine) as described in Materials and
Methods. Valine was then added to induce isoleucine limitation. Valine and
isoleucine were added to a parallel culture as a control. RNA was isolated
from cells prior to and at 7 .5, 15, 30 minutes after the addition of valine. Sl
assays were performed as described in Materials and Methods with the 5’
end-labeled EcoRV fragment as a probe. Positions of transcripts from man
promoters are indicated. Size markers are denatured HpaII restriction

fragments of pUC 19.

158

Ile

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'LQGQ'W
Time(min) ONE SHE.) 8
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Vol

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gg‘IZ—————~-—v-probe
242-. _ ..

 

. ..rpoH3p

 

DISCUSSION

Sigma factors confer upon core RNA polymerase an ability to recognize a
specific group of promoters. The roles of alternate sigma factors in expression
of SPOl genes during its propagation or in uninfected B. snhtilis are well
studied examples (9, 24, 25). Four sigma factors encoded by the E. 99.11
genome and distinguished by size have been identified. a” encoded by the
mall gene is the major sigma factor. a"2 encoded by 232211 (111323) confers
recognition of heat shock promoters (8, 15, 22). a“ encoded by IRON (ntrA,
glnE) is required for expression of genes involved in nitrogen utilization (18,
19). The fourth, a" of 28 KDa, controls transcription of flagellar and
chemotaxis genes (2). We have identified a fifth sigma factor of 24 KDa which
confers recognition of one of the mall promoters, 3P. Antibodies raised
against a synthetic peptide corresponding to the most highly conserved region
of bacterial sigma factors were used in Western-blot analysis of whole cell
lysates of E. 9911 (11). These experiments resulted in cross-reactivity with 67",
a", and proteins of 75, 27, and 23 KDa. We presume that the latter two
proteins correspond to 0" and 0". Identification of other genes transcribed by
this form of a“-RNA polymerase is important in understanding the role of
this sigma factor in the growth of E. £911- A consensus promoter sequence
derived from a sequence analysis of promoters recognized by E0" may reveal

how different holoenzymes recognize their respective promoters.

159

an“

160

The expression of the man gene appears very complex. mHlP and 4P
are transcribed by Ed” (10) while mH3P is transcribed by Ea" as shown
here. 1:991:12? appears to be strain specific. Second, dnaA protein involved in
initiation of DNA replication regulates expression from 3P and 4P (35).
Third, upon temperature upshift, mH. transcript levels increase with 2P and
3P increasing the most (10). Fourth, a downshift in temperature also results
in increased levels of mHlP and 3P mRNA (unpublished results). The
variety of treatments such as an increase in temperature, ethanol,
bacteriophage infection, and others (reviewed in 1) which induce expression of
heat shock proteins may occur by different mechanisms resulting in increased
transcription from one or more of the mH promoters.

Expression of heat shock proteins is also induced by the stringent
response (16). This condition also results in synthesis of stringent starvation
protein comprising at least half of the proteins synthesized (29). We
' considered whether a“ and stringent starvation protein were identical. The
observation that mgH3P transcription was not elevated but diminished to
undetectable levels under stringent response conditions indicates that a“ and
stringent starvation protein are not the same. That mH transcripts were not
elevated by this treatment also suggests that stringent induction of heat
shock proteins does not require a” but an alternate sigma factor.
Alternatively, stringent induction may not act at the transcriptional level of
3221-1 expression but past-translationally by stabilization of a”.

161

Note: While these studies were being completed, J .W. Erickson and CA.
Gross reported to have identified a novel sigma factor of 24 KDa which
promotes transcription from mH3P at the Molecular Genetics of Bacteria and
Phages Meeting, Cold Spring Harbor, N.Y., August, 1988.

10.

11.

13.

14.

15.

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Bloom, M., Skelly, S., VanBogelen, R.A., Neidhardt, F.C., Brat, N., and
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Bradford, M.M. (197 6) Anal. Biochem. 72, 248-254.

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Burgess, RR, and Jendrisak, J.J. (1975) Biochem. 14, 4634-4638.

Cashel, M. and Rudd, KE. (1987) Emhendua 9911 and Salmonella
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Cowing, D.W., Bardwell, J.C.A., Craig, E.A., Woolford, C., Hendrix,
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Doi, RH., and Wang, L.-F. (1986) Microbiol. Rev. 50, 227-243.

Erickson, J .W., Vaughn, V., Walter, W.A., Neidhardt, F.C., and Gross,
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Fujita, N., Ishihama, A., Nagasawa, Y., and Ueda, S. (1987) Mol. Gen.
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Fajita, N., and Ishihama, A. (1987) Mol. Gen. Genet. 210, 10-15.

Gill, D.R, Hatfull, G.F., and Salmond, G.P.C. (1986) Mol. Gen. Genet.
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Grossman, A.D., Taylor, W.E., Burton, Z.F., Burgess, RR, and Gross,
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Hager, DA, and Burgess, RR (1980) Anal. Biochem. 109, 76-86.

Hirschman, J., Wang, P.K, Sei, K, Keener, J ., and Kustu, S. (1985)
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Hunt, T.P., and Magasanik, B. (1985) Natl. Acad. Sci. USA 82,
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Ishihama, A., and Saitoh, T. (1979) J. Mol. Biol. 129, 517-530.
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164

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Chapter V
PREFERENTIAL BINDING OF dnaA PROTEIN TO DNA FRAGMENTS
CONTAINING PROMOTER REGIONS OF mlA, m3, AND and,
AND TO FRAGMENTS CONTAINING SOME UNIDENTIFIED SITES

165

ABSTRACT

Binding activity of dnaA protein to DNA fragments containing the
promoter region of mlA. m3, or nnl was tested by nitrocellulose filter
binding assays. In comparison to fragments containing the dnaA promoters or
the pBR322 origin, the nolA, m3, and nrd fragments were retained with
greater afinity. Filter binding assays were also performed with a restriction
enzyme digest of chromosomal DNA. Some unidentified fragments were
preferentially bound. A cloned EcoRI-HindIII fragment of about 700 bp was
bound by dnaA protein more strongly than the dnaA promoter fragment.

166

INTRODUCTION

dnaA protein is an essential protein for initiation of DNA replication from
the chromosomal origin of E. coll (1-3). It binds to the mic region and
functions in an early stage of initiation (4). dnaA protein also binds to other
DNA fragments containing a consensus sequence of 'I'I‘AT(A/C)CA(A/C)A (5).
Among these are the dnaA promoter fragment and a fragment containing the
promoter region of a gene encoding a 16 KDa protein. The binding at those
promoter regions results in transcriptional repression of these genes by dnaA
protein (6-9).

In addition to E. coll genes described in previous chapters, the dnaA
protein binding sequence is present in regulatory regions of several other
genes including polA, m3, and nnd, (10-14). mlA is the gene coding for the
DNA polymerase I (11). The product of the m3 gene is a subunit of the
uvrABC endonuclease (18). Both uvrB protein and pol I are involved in DNA
repair. Ribonucleotide reductase which catalyzes the conversion of
ribonucleotide to deoxyribonucleotide is encoded by the nrdAE operon (13).

The presence of dnaA binding sites in these regulatory regions prompted
us to test the influence of dnaA protein on the expression of these genes.
Experiments described in this chapter indicate that dnaA protein binds to
promoter regions of these genes. However the efl'ect of binding was not

conclusively determined.

167

168
Since only a limited portion (about one fourth) of the E. coli genome is
currently sequenced, efi‘ort was also made to isolate DNA fragments bound by
dnaA protein from a chromosomal DNA digest in attempt to identify
unsequenced genes controlled by dnaA protein. Although this approach did
not result in any positively identified genes, it did show that certain DNA
fragments are preferentially bound by dnaA protein.

MATERIALS AND METHODS

Wm

E. coli W3110 (wild type) was from the E. coli genetic stack center.
Chromosomal DNA for binding assays was prepared from W3110 as described
(16). Plasmid pCJ 60 containing the 592 bp HpaII fragment of the mlA
promoter region was obtained from Drs C. Joyce and N. Grindley (Yale
University) (17). pDR1494, from Dr A. Sancar (Yale University) (18), contains
the 6.5 kb PstI fragment of the m3 gene and its promoter region. Plasmid
pPSZ, carrying the 12 kb PstI fragment of the nnl gene and the promoter
region, was from Dr. B. Sjoberg (Swedish University) (19). All three plasmids
are pBR322 derivatives. The plasmid pUVRB is a recombinant of pKOl (22)
constructed by cloning the m3 promoter region contained in a 600 bp Tan
fragment at the SmaI site. A shorter fragment (1.6 kb EcoRI-KpnI) containing
the mzd promoter region was subcloned into pUC 19, resulting in pNRD.
Plasmid DNAs and DNA fragments were prepared as described in previous
chapters.

Restriction endonucleases and dnaA protein are from the same sources as

described in chapter IV.

III I] I fill I. 1'
Binding assays were performed as described in previous chapters. Assays

were performed with 100 ng plasmid DNA digest or with 0.025 pmol of

169

170
purified DNA fragments. 10 pg of EcoRI digested chromosomal DNA was used
where as indicated. DNA fragments were end-labeled either at 3’ end or at 5’
end. The labeling was performed as described in previous chapters. The

amounts of dnaA protein used were as indicated.

RESULTS

00:; 00‘! (‘OQOHOOO i'0 ‘0 '5 .0 00000: ”400:0 001‘» I: 3.00
and

Three copies of the dnaA protein binding sequence (dnaA box) (one 9/9
and two 8/9 matches) are present at 232-253 and 374-383 bp upstream of the
coding sequence of the nolA gene (Figure 1A) (11). Since transcriptional start
sites have not been mapped, positions of these dnaA boxes relative to
promoters cannot be determined. In the case of MB, transcriptional start
sites have been mapped at three positions (Figure 1B) (12). The two dnaA
boxes (one 9/9 and one 8/9 match) are located at 22-45 bp upstream of the 3P
RNA start site. One copy of dnaA recognition sequence (9/9) is found at 43-52
bp upstream of the only RNA start site of the nnl gene (Figure 10) (13).

3.1. Ell l'lllll I .
The binding activity of dnaA protein to promoter regions of 991A, math,
and nrd was tested by nitrocellulose filter binding assays. Plasmid pCJ 60,
which contains the 592 bp HpaII fragment of the pole. regulatory region in a
pBR322 derivative, was digested with HpaII and used in binding assays
(Figure 2). Two fragments were retained as increasing amounts of dnaA
protein were added, one about 527 bp containing the pBR322 origin and the
other about 1.2 kb (markers not shown). One dnaA box is present near the

replication origin of pBR322. DNA fragments containing the origin were

171

 

172

Figure 1. Map of the promoter regions of polA, math, and nzd.

(A) polA. The coding region is indicated by a cross hatched box. The
dnaA recognition sequences (dnaA boxes) are the stippled boxes. The arrows
indicate the relative orientation of the dnaA boxes. The numbers are positions
of the dnaA boxes relative to the initiation codon.

(B) M. Relevant restriction sites are as indicated. Promoters are
indicated by arrow boxes. Positions of the dnaA boxes are compared to the 3P
mRNA start site. Other designations are as in (A).

(C) nnl. Designations are as in (B).

 

173

 

 

 

 

A.
Hoall Hpall
F l l l l l l
o 200 400 600 no
-> -D- ->
E] In E] W
(3233-374) (2515-232) DolA
dnaA boxes
8.
Taql Hpall Hpall EcoRI Taol
I I I I I I I
o 200 400 600 bp
Ip C2+
2p ELI-V
32:. '
El E! W
(45-22) uvrB
dnaA boxes
Konl EcoRV EcoRI
I fl I I I r I fi
o 500 l000 , ’ Isoo bp'
, ’ I
I
, , ’ I
, ’ I
I
I ’ |
, ’ I
/ ECORI
x ’ I I
I I I I I I I
0 I00 200 300 bp
2” ,
,, pm, W
[233
<52-43) ”rd

dnaA box

174

Figure 2. Preferential binding of dnaA protein to a restriction fragment
containing the polA regulatory region.

Filter binding assays were performed as described in Materials and
Methods with the indicated amounts of dnaA protein and 100 ng of 3’ end-
labeled pCJ 60 digested with HpaII. TD, total DNA digest; B, bound fragments
eluted from the filter; F, fi'agments which flowed through the filter. The
positions of DNA fragments containing the pBR322 replication origin or the
regulatory region of the nolA gene are as indicated.

 

 

 

175

100 dnaA protein (ng)

50
I__I
BF
II

25

0

Fl
Bl

EH

mm...

D
Tl

“---—- ~.- -.-pOIA

: A I: 1 g f-pBR322 orI
. .

u
a“.

 

 

 

176
observed previously to be bound by dnaA protein with a similar aflinity as
that of the dnaA promoter fragment. The other retained fragment of about 1.2
kb contains the cloned nolA sequence and part of the pBR322 sequenceua 622
bp HpaII fragment of pBR322. Since the 622 bp vector fragment was not
bound by dnaA protein (chapter 2, Figure 2), the binding affinity of the 1.2
kb fragment must have originated from the nolA sequence. The relative
amounts of the two fragments in the bound lanes indicate that the binding
afiinity to the polA fragment is similar to or greater than that to the pBR322
origin fi'agment.

The M promoter region is contained in pDR1494. pDR1494 was
digested with Tan and the digest was used in filter binding assays (Figure
3). Two fragments were retained: one 1.3 kb containing the pBR322
replication origin; the other 600 bp presumably containing the mmB, promoter
region. The bound DNA was further digested with EcoRI to confirm that the
retained fragment contains the m3 promoter region. Only two EcoRI sites
are present in pDR1494, one in a 368 bp vector fragment, the other in the
m3 promoter region (18). The 600 bp fragment was cleaved to a shorter
fragment, indicating that it contains the 1133 promoters. To further compare
the binding affinity of the m3 fragment to the pBR322 origin fragment,
assays were performed with HpaII digest of pUVRB, which contains only the
600 bp Tan fragment in pKOl (a pBR322 derivative) (Figure 4). A 400 bp
fragment was retained in addition to the 527 bp pBR322 origin fragment
(Figure 4A). A fragment from the vector pKOl coelectrophoresed with the 400

bp fragment. This vector fragment was not bound by dnaA protein as shown

F”)

177

Figure 3. Binding of dnaA protein to restriction fragment containing the m3
promoter region.

Filter binding assays were performed as described in Materials and
Methods with 100 ng of dnaA protein and 100 ng of 3’ end-labeled pDR1494
digested with Tan. B, bound fragments eluted from the filter; F, fragments
which flowed through the filter. EcoRI, DNA fragments eluted fi-om the filter
was firrther digested with EcoRI. Positions of DNA fragments containing the

m3 promoter region or the pBR322 replication origin are as indicated.

 

 

 

178

EcoRI

.4.

i i3 i
----= —pBR322 orI'

 

...: . :- 3. x

179

Figure 4. Preferential binding of dnaA protein to the m3 promoter region.

(A) Filter binding assays were performed as described in Materials and
Methods with 100 ng 3’ end-labeled pUVRB digested with HpaII. The
amounts of dnaA protein used are as indicated. Positions of the pBR322
origin fragment and the m promoter fragment are also indicated. TD, DNA
digest; B, bound fragments eluted from the filter; F, fragments which flowed
through the filter.

(B) Binding assays were performed with 3’ end-labeled pKOI digested
with HpaII. Other designations are as in (A).

 

180

 

 

35
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£395 <96 00

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mm>3a

181
in parallel assays performed with pKOI (Figure 4B). The amounts of the two
fragments in the bound lanes indicate that the 400 bp m3 fragment was
bound with greater affinity than the origin fragment.

Binding assays were performed with a purified DNA fragment containing
the nnl promoter region (Figure 5). A 511 bp EcoRV-EcoRI nnd fragment was
mixed with equal molar amounts of DNA fragments containing the dnaA
promoter (945 bp), the mH promoter (342 bp), and the lacUV5 promoter (203
bp). As described in the preceding chapters, the moH promoter region was
bound by dnaA protein with higher affinity than that of dnaA promoter
region. The lacUV5 fragment was poorly bound by dnaA protein and used
here as a negative control. The binding afi‘inity of the and fragment appears
to be greater than that of the dnaA promoter fragment, but less than that of
the [pol-l fragment.

Transcription run-off assays were performed in attempt to assess the

effect of binding. However the results were circumstantial and inconclusive.

 

To date, about one fourth of the E. coli genome is sequenced. In order to
obtain DNA fragments bound by dnaA protein in the unsequenced portion of
the genome, filter binding assays were performed with chromosomal DNA
digested with EcoRI (Figure 6). The assays were with 10 pg of radioactively
labeled DNA and a limiting amount of dnaA protein (260 ng) to obtain
fragments bound with high affinity. About 1% of the total input was retained
on the filter. DNA fragments retained were separated on an agarose gel

182

Figure 5. Binding affinity of dnaA protein to DNA fragment containing the
m promoter region.

Binding assays were with 0.025 pmol each of DNA fragments containing
the promoter region of M moH, nrd, or law. DNA fragments were 5’
end-labeled. The amounts of dnaA protein used are as indicated. TD, the
mixture of DNA fragments used in the assay; B, fragments eluted from the
filter; F, fragments that flowed through the filter.

_ ww- ‘-

183

0 100 150 200
F_l r—1 r-1 r—1
BIPIFFFPE

   

dnaA
protein (ng)

  

184

Figure 6. Preferential binding of dnaA protein to chromosomal DNA
fragments.

Binding assays were performed with 10 pg of chromosomal DNA digested
with EcoRI and 260 ng of dnaA protein. B, fragments eluted from the filter;
F, one tenth of the fragments that flowed through the filter.

 

185

dnaA protein (ng)

260

 

1.2 kb

my»... m m...
x
maxim, Ears ..

186
together with one tenth of the DNA in the flow through. dnaA protein
preferentially retained several fragments, most obviously a fragment of about
1.2 kb.

Since none of the known binding sites are expected in a 1.2 kb EcoRI
fragment (20, 21, 10-13), the fragment retained from the chromosomal DNA
must represent a new binding site. To facilitate further characterization, this
fragment was purified from the bound DNA after electrophoresis, and a 700
bp EcoRI-HindIII subfragment carrying the binding site (data not shown)
was cloned into M13mp18. Binding assays were performed with purified
fragments. The 700 bp EcoRI-HindIII fragment was mixed in equal molar
amounts with the dnaA promoter fragment (945 bp) and the 31911 promoter
fragment (342 bp). The binding affinity of the cloned 700 bp fragment is
between that of the mall and dnaA fragments. The minor band below the
cloned fragment may be a deletion product. This fragment was not bound well
by dnaA protein presumably because the binding site has been removed.
Initial efforts in cloning the 700 bp fragment into plasmid vectors (pBR322
and pUC 19) were unsuccessful, presumably because the presence of the 700
bp chromosomal fragment in high copy number is deleterious to cell growth.
Similar lethal efi‘ect has been observed with the m3 promoter region (23).
This may be due to titration of dnaA protein from the essential sites in the
E. coll chromosome. Further effort in characterization of this fi'agment was
unsuccessful. Sequencing of the fragment may be required for any further

experimentation.

187

Figure 7. Binding amnity of a 700 bp cloned fragment.

Binding assays were performed with 0.025 pmol each of 5’ end-labeled
DNA fragments containing the promoter region of dnaA or mil, and a
fragment containing the 700 bp cloned chromosomal fragment. The amounts
of dnaA protein used are as indicated. TD, the mixture of DNA fragments
used in the assay; B, fragments eluted from the filter; F, fragments flowed
through the filter.

188

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(NCO I I'll-Ill. .l l l

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355303 <95 com 02 om mm o

DISCUSSION

Experiments presented here indicate that dnaA protein binds to promoter
regions of genes pglA, um, and and. Several unidentified chromosomal
fragments are also preferentially bound by dnaA protein. The binding
affinities are comparable to the binding at the dnaA promoter region which
results in transcriptional repression of the dnaA gene. This may suggest the
relative importance of these binding events.

Preliminary results of in vitro transcription run-ofl‘ assays indicate that
transcription from promoters of the polA gene and of the m3 gene may be
inhibited by dnaA protein. However, effort in detection of transcripts from
these promoters in viva was not successful due to technical difliculties. The
possible regulation of nolA and m by dnaA protein suggests that the DNA
replication process could be correlated with DNA repair. Transcription from
the nnl promoter seems not to be influenced by dnaA protein in vitro. Since
inhibition of transcription by dnaA protein may occur by promoter occlusion,
the lack of repression on the nnl promoter could be due to the distance
between the dnaA binding site and the promoter. Alternatively, other factors
required for any repressive (or stimulatory) effect may be absent in the run-
off assays. .

A current computer analysis of about 1000 kb of the sequenced portion of
the E. coli genome yielded 27 sites of 'I‘I‘AT(A/C)CA(A/C)A (Table 1). Among
them are 8 copies of 'I'I‘ATCCACA (Table 2), 8 of TTATACAAA (Table 3), 9 of

189

190

Table 1. Number of sites of the dnaA binding sequences

in the sequenced g. coli genome.

 

 

 

Number of Coding Regulatoryh

Sequences‘ Sites Region Region Others
TTATCCACA 8 1 7
TTATACAAA 8 4 4
TTATCCAAA 9 4 5
TTATACACA 2 1 1°
a Only the four sequences are listed.
b The binding sequences are within 506 bp upstream of

initiation codon or within 137 bp upstream of mRNA start

site.

c This binding site is present in a bent DNA fragment (22).

191

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192

 

 

 

 

 

 

 

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Ame. 4N- «Ammv ma: oncoamom

OH «Ammv mH+ xxmflv soa+ xoonm amom «masons mmmm

an xoecc «ma: mnmonucsm «2o H ammnoesaom <29 «How

mm Amumncson sumac mm~+ conomonacom «2o onuo

mm .xsamncson sumac mm+ nonsmonacom <28 anao

ammuosvcm

ma fizmv va mamonucmm mezc cpfiuocaosconfim cu:

mm Awedv mom+ Emflaonmucz manusm cmmccooncanco mzH mmsm
coaumasmom meow

em Ammc Hm- rxmav m~+ «nonsmonaaom «2a anoaoaa «man «was

.mmm .COflumooq coauocsm cwmuoum meow

 

.mwuflm ¢U<UUB¢BB MO mcoflumooq .N mamas

 

193

 

 

 

 

 

 

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.N canon CH confluence mm cum mcoflumcofimoo m

em .«zmn mmc mmm+ mzmu conodo «zmu menu

mm x0948 H~m+ cofimao>cH mza ammuuo>cH cza mmm

mm fismv mmI cowumafisflmmd cooonufiz ammuoscom cumnuflz mnflc

18:..va oml 222$: NI mammnucmm

Hm rx-zmmv mNHI.xx-:mflv Hm- uno< oasec commoumom can moan

om Awe¢v NvHI unommcmua cuoH cccw anoumasmwm conH mmw

mm xoa<c em- coHumonuneoe 42mg ommaanaoe «zmn mm mmamm

unommcmns cmmccooucanao III

mm noes. mmmfl+ nfioc acne< cam amosm mamaomauo can
mammnucam cmmucmmcmnuaaoemnumu

hm Aoeav m+ peed ocaE¢ meanuficno mmmm

.mom coaumooq coauocnm cacuonm comm

 

.mmuflm <4¢U<B<BB Mo mGOflumooa .m

magma

194

.N canmu ca ccnwnomcc mm mum occaumcmemoo m

 

 

 

 

 

 

Nv ADB<V omHI unommcmna mmoamx cacuonm maxx mwNm

Emaaonmumz cmmcmmouc>nco III

as Aware m+ nnoc ocHe< acacooone nun
cmmaaemnumomcmna

ow rams ovm- rxmfic FNH- mnmonacmm manonEnusm oumaamcmm mmwm

mm isnmccson sumac smH+ nonsmonacom «2o once

mm Aoa<v Hmm+ Emfiaonmucz amouamz cwcuonm Same Hams

mm Ammv vaI «Away mm- conamanEnmmc comoaanz ammuonacsm madamasao «mam

conwao

em xoacv man- crocxco czocxcs oaoc

mm AcoHomanHoav H+ mammnacsm unanaoncmonm ommcnx onnooosamna mam

mm Aweav mHHI czocxcs Camuoum Umc mUwU

.mcm cofiumooq cowuocsm cacuonm coco

 

.moanm 4<<ooe<ea no mconamooq .v manna

195

Table 5. Locations of TTATACACA sites“

 

 

Gene Function Location Ref.
bentS unknown bending site 22
oriC DNA replication +200 (left boundary) 26

 

a Designations are as described in table 2.

196
TTATCCAAA (Table 4), and only 2 of TTATACACA (Table 5). The number of
sites of the former three sequences is in good agreement with the expected
frequency—31 sites each in the 4000 kb genome. The frequency of the fourth
sequence is lower than the expected. The reason for this is unknown. Analysis
of locations of the sequences 'I'I‘ATACAAA and 'I'I‘ATCCAAA indicates that
they are distributed about equally in coding regions and in regulatory regions
upstream of open reading frames. On the other hand, all copies of
'I'I‘ATCCACA but one are in regulatory regions, including two in the mo
region. The difference in location may suggest that the sequences
'I'I‘AT(A/C)CAAA are less significant functionally than the sequence
'I'I‘ATC CACA so that the latter is preferentially selected in promoter regions
through evolution whereas the distribution of the former is rather random. In
another word, the sequences 'l'l‘AT(A/C)CAAA are less well bound than
'I'I‘ATC CACA. In supporting this conclusion, a TTATACAAA site within M13
genome and a TTATCCAAA site in the promoter region of the gln gene
(encoding glutamine synthetase in nitrogen assimilation) are poorly bound by
dnaA protein (5, data not shown).

Transition of A to T at the third position of the nine base-pair dnaA
protein binding sequence does not seem to significantly influence the binding
afiinity in the case the 16 KDa gene. One 'I'I'I'I‘CCACA site in the promoter
region of the 16 KDa gene has a similar binding aflinity compared to the
dnaA promoter region which contains a 'I'I‘ATCCACA site. The binding to
either promoter regions results in transcriptional repression to a similar
degree. However, a 'I'I‘TTCCACA site in the coding region of the dnaA gene

197
appears to have much lower afinity than the site in the dnaA promoter
region. Sequences outside the nine base-pair may also afi'ect the binding
activity. The exact sequence requirement for dnaA binding awaits further
experimentation.

Among the 8 'I'I‘ATCCACA sites, two are in the mo minimum sequence;
one copy is at each promoter region of dnaA, mall, 991A, m3, and 11:11; and
one is in the coding region of the M gene coding for IMP dehydrogenase
in the metabolism of purine (Table 2). All these sites were shown to be bound
by dnaA protein except the site in mm which was not tested. The binding
resulted in functional involvement of dnaA protein in initiation of replication
and expression of dnaA and mall gene. Further experiments are needed to
clarify the role of dnaA protein in expression of mlA, maB, and nul.

Binding assays with chromosomal DNA fragments indicate that there are
some unidentified binding sites on the chromosome. A total of 31 dnaA
recognition sequence TTATCCACA are expected in the 4000 kb genome, not
including the other three variants. This seems to be a large number for a
regulatory protein. The location of the binding sites may be critical for
functional involvement, which may reduce the number of efl'ective binding
sites. On the other hand, dnaA protein may regulate a large number of genes.
The level of dnaA protein is expected to be constant independent of growth
rate due to autoregulation of the dnaA gene. As a result, the concentration of
proteins under its control will also be kept constant. Many E. coli proteins
may have a relatively fixed concentration independent of growth rate. These
genes may be potential candidates to be regulated by dnaA protein. The exact

198
function of dnaA protein in regulating gene expression awaits further

exploration of genes under control of dnaA protein.

.51

S9935.“

10.

11.

13.

14.

15.
16.
17.
18.

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10856-10861.

Bognar, A. L., Osborne, C., and Shane, B. (1987) J. Biol. Chem. 262,
12337-12342.

Reitzer, L. J., and Magasanik, B. (1985) Proc. Natl. Acad. Sci. USA 82,
1979-1983.

Cole, S. T., and Raibaud, O. (1986) Gene 42, 201-208.

Tumbough, C. L., Hicks, K L., and Donahue, J. P. (1983) Proc. Natl.
Acad. Sci. USA 80, 368-372.

Aronson, B. D., Ravnikar, P. D., and Somerville, R L. (1988) Nucl.
Acid Res. 16, 3586.

Davis, E. O., and Henderson, P. J. E. (1987) J. Biol. Chem. 262, 13927
13932. .

Chapter VI
SUMMARY AND PERSPECTIVES

202

SUMMARY AND PERSPECTIVES

dnaA protein binds to mo and functions in initiation of DNA replication.
It also binds to promoter regions of its own gene, the 16 KDa protein gene,
mH, mlA, uvrB, and nnl. The binding results in transcriptional repression
of the former three genes, and the efi'ect on the later three are not
determined. Autoregulation of the dnaA gene provides a relatively constant
concentration of dnaA protein, so that its accumulation is proportional to the
increase in cell volume, that is the growth rate. Assuming initiation occurs
when a certain amount of dnaA protein has accumulated, initiation of
replication can then be kept in pace with cell growth. Transcription fiom the
16 KDa promoter seems to influence initiation from mg. Regulation of
transcription from the 16 KDa promoter by dnaA protein may directly
contribute in regulation of the initiation event.

Regulation of the mall expression appears to indirectly influence its
activity in DNA replication. The moH gene encodes a sigma factor, sigma32,
that recognizes promoters of heat-shock genes. The level of sigma32 controls
levels of heat-shock proteins. Heat-shock proteins, in turn, seem to modulate
the replication activity of dnaA protein. Both in vitro and in viva evidence
indicates that dnaK protein is involved in this process. dnaK protein was first
observed to stimulate the activity of purified dnaA46 protein in vitro (Hwang
and Kaguni, unpublished results). The wild type dnaA protein also seems to
be modified by dnaK protein (Carr and Kaguni, unpublished results).
Consistent with these observations, a dnaK mutant has been obtained which

203

204
is defective in initiation of replication presumably because the unmodified
dnaA protein is not as active in supporting initiation (1). groEL and groES
proteins also appear to interact with dnaA protein in vivo (2, 3). Mutations in
the mall gene result in inhibition of cell division, possibly caused by the
defect in initiation (4).

A regulatory circuit between dnaA protein and the heat-shock system is
summarized as follows. dnaA protein regulates expression of heat-shock genes
through [ml-I. In turn, heat-shock proteins modulate the activity of dnaA
protein. A balance between dnaA protein and the heat-shock proteins might
be maintained at a specific level in cells grown at a fixed temperature.
Regulation by dnaA protein provides a constant concentration of heat-shock
proteins under normal growth conditions. On the other hand, conditions
detrimental to cell growth might reduce the activity of dnaA protein, blocking
DNA replication, and simultaneously increasing synthesis of heat-shock
proteins, the function of which has been known as stress coping. One such
example is the SOS response. Transcription from the dnaA promoters
increases significantly after induction of the SOS response, indicating that the
level of active dnaA protein as a repressor is reduced (5). A difl'erent pathway
of DNA replication independent of dnaA protein is induced (6). Synthesis of
heat-shock proteins is also induced (7 ). These observations fit well into a
picture where dnaA protein is inactivated during the SOS response. If dnaA
protein represses expression of polA and m3, inactivation of dnaA protein
could induce the synthesis of pol I and uvrB protein, both of which are
involved in DNA repair. Further proof of inactivation of dnaA protein will

205
support this model. Identification of new genes controlled by dnaA protein will

also provide insights into the role of dnaA protein in the process of cell
growth.

206

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Jenkins, A. J., March, J. 3., Oliver, I. R, and Masters, M. (1986) Mol.
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Fayet, O., Lourarn, J .-M., and Geargopoulos, C. (1986) Mol. Gen. Genet.
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Tsuchido, T., VanBogelen, R A., and Neidhardt, F. C. ( 1986) Proc.
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Quinones, A., Kaasch, J., Kaasch, M., and Messer, W. (1989) EMBO J.
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Walker, G. C. (1984) Microbiol. Rev. 48, 63-93.

Krueger, J. H., and Walker, G. (1984) Proc. Natl. Acad. Sci. USA 81, ~
1499-1450.