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Detenmination of the Rabbit a and a Globin Nascent
Polypeptide Size Distribution: Correlation of Nascent
Peptide Accumulations with mRNA Secondary Structure

By

Calvin P. H. Vary

A Dissertation
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry
1980

ABSTRACT

Determination of the Rabbit a and 8 Globin Nascent Polypeptide
Size Distribution: Correlation of Nascent Peptide
Accumulations with mRNA Secondary Structure

by
Calvin P.H. Vary

Polysome derived rabbit globin nascent polypeptides were isolated and
the size distribution of the a and B globin nascent polypeptide components
was determined. The size distributions of the a and 3 globin nascent poly-
peptides were shown to be non-uniform and unique. Previous work (1) has
established a correlation between the accumulation of certain size classes
of nascent polypeptides and the presence of elements of secondary structure‘
in a mRNA molecule which, we propose, hinders ribosomal transit in certain
regions of the mRNA molecule giving rise to the observed nascent polypep-
‘tide size non-uniformity. ,

Globin nascent polypeptides were prepared by isolation of peptidyl tRNA
from polysomes which had been labeled to steady state with a particular
Icadiolabeled amino acid. Following base catalyzed hydrolysis of the tRNA--
nascent peptide linkage, the size distribution of the nascent polypeptides
was determined by analytical gel chromatography. The a globin polypeptide

size diStribution was determihed by labeling polysomes in a reticulocyte

ceH '

cell free protein synthesizing system derived from rabbits homozygous for
Ithe 3112 isoleucine. valine polymorphism. Such a lysate was shown to
incorporate radiolabeled isoleucine into a globin precursors only since the
B globin molecule contains no isoleucine residues.

The B globin component of the globin nascent polypeptide size distribu-
tion was determined by fractionation of tryptophan labeled nascent peptides .
according to size, treatment of various fractions with trypsin and separa-
tion of the two 8 and one a tryptOphan labeled tryptic peptides by high
pressure liquid chromatography followed by quantitation by liquid scintil-
lation spectrometry. ' I

The resolved 3 globin nascent peptide size distribution was found to be
non-uniform and substantially different fran the size distribution of the a
globin nascent polypeptides. The 8 globin nascent peptide size distribu-
tion was compared to a primary and secondary structural model of the B glo-
bin mRNA. Nascent peptide accumulations were found to correlate with pre-
dicted regions of stable secondary structure (2) providing a correlation

betWeen the secondary structure of a mRNA in vivo and the secondary Struc-

 

ture of the same mRNA determined with the aid of Chemical probes in 11359.
The a globin nascent polypeptide size distribution was used to deter-

mine if agents which can modify RNA secondary or tertiary structure would
alter the non-random distribution of ribosomes along the mRNA as reflected
-lll changes in the size distribution of nascent a globin polypeptides.
'Thermal perturbation was effected by temperature shift experiments between
15 and 37°C following steady state labeling of polysomes. 'Such experiments
revealed a moderate and reproducible shifting of discreet components of the

a globin size distribution as a function of temperature under conditions

which Span 10-15% of the total thermal hypochromic shift of the globin mRNA
molecules.

Perturbation experiments were carried out with a short mRNA complimen-
tary oligodeoxyribonucleotide. The reticulocyte lysate was incubated in
the presence of 100 pMolar tetradeoxycytidylic acid which is complimentary
to three positions in the 8 globin mRNA and has no complimentary sequences
in the a globin mRNA molecule. Tetradeoxycytidylate had little effect on
the a globin nascent peptide size distribution but caused shifts in dis-
creet regions of the combined a plus 8 nascent polypeptide size distribu-
tion. This is consiStent with tetradeoxycytidylate interaction with the 3
globin mRNA molecule resulting in a perturbation of the mRNA structure
which was effectively monitored in solution by the redistribution of the
nascent polypeptide size distribution.

Further experiments involving the effects of the elongation inhibitors
cycloheximide, gougerotin and sparsomycin on the a globin nascent peptide
size distribution were conducted. It was found that ribosomal loading of
the mRNA produced by all the above elongation inhibitors resulted in the
reproducible relative loss of high molecule weight nascent polypeptides of
the approximate size of completed a globyl tRNA. Similar results were
observed when polysomes were fractionated by sucrose density gradient sedi-
mentation. The size distribution of a a globin nascent polypeptide deter-
mined as a function of polysome size for both sparsomycin inhibited and
control polysomes all revealed diminishing amounts of completed and near
completed globyl chains in larger polysomes relative to smaller length oli-
gopeptides. TheSe results are consistent with the facilitated release of
completed globyl polypeptides from larger polysomes indicating possible

ribosomal relaxation of mRNA conformation in or near the termination codon.

1.

2.

Chaney, N.G. and Morris, A.J. Archs. Biochem. Biophys. 125, 283-291
(1979).

Vary, C.P.H., Pavalakis, G.N., Vournakis, J.N. and Morris, A.J. Nature
(1981) in Press. '

TABLE OF CONTENTS

 

Introduction
RNA Primary Structure ...................... 1
RNA Secondary Structure ..................... 1
Optical Properties of RNA Helices ................ 3
Thermodynamic Properties of RNA Helices ............. 5
Experimental Approaches to RNA Structural Analysis ....... 5
Secondary Structure and RNA Function .............. 9
Rabbit Globin mRNA, Sequence and Translation ......... 11
mRNA Translational Dynamics .................. 13
me ............................. 18
Methods
Preparation of Rabbit Reticulocytes .............. 21
Preparation of the Reticulocyte Lysate ............ 21
Conditions for Cell-free Protein Synthesis ........... 21
Cell-free Globin Biosynthesis: Confirmation of Product
Identity, Separation of a and a Globin Chains ....... 22
Preparation of Peptidyl tRNA - Polysomal Labeling ....... 22
Dissociation of Polysomal Structures .............. 23
Purification of Peptidyl tRNA ................. 23
Recrystallization of Guanidinium Chloride ........... 25
Analysis of Peptide Size Distribution ............. 26
Preparation of Cyanogen Bromide Fragments of Labeled a
and B Globin. . . . . ................... 27
Determination of Distribution Coefficients ........... 28

Identification of 3112 (Val/Val) Homozygous Rabbits ...... 29

8y Rapid Estimation of L-Isoleucine Incorporation SDS-gel
Electrophoresis ................. . . . . . 30

Preparation of Uniformly Labeled L-[14CJ-Trp-Globins . . . . . 32

Tryptic Peptide Analysis of L-[3HJ-Trp Labeled Nascent
Peptides .......... . . ....... . ..... 32

Tryptic Digestion of Nascent Peptides ..... . ....... 32
Preparation of Bio-gel P-2 (-400 mesh) Column Buffer . . . . . 33
Preparation of Bio-gel P-2 (~400 mesh) Analytical Column ... ., 34

Bio-gel P-2 (-4DO mesh) Chromatography of Tryptic Digestion
Products ......................... 34

Construction of the High Pressure Liquid Chromagraphic System.. 35

Preparation of Buffers for High Pressure Liquid
Chromatography ...................... 35

High Pressure Liquid Chromatographic Analysis of Tryptic
Digestion Products .................... 36

Qualitative Analysis of Tryptic Digest Products -
Desalting of Bio-gel P-2 Digestion Products ........ 37

Identification of Resolved Tryptic Digestion Components . . . . 37

Paper Chromatographic Analysis of Resolved Tryptophan-
Labeled a Globin Tryptic Digestion Products ........ 38

Calibration of the Bio-gel P-2/Urea Chromatographic
System for Double Isotope Analysis ...... . ...... 38

Calibration of the HPLC System for Double Isotope Analysis . . 39

Separation of the Different Polysomal Size Classes by

Sucrose Density Gradient Centrifugation .......... 40
Results
Analysis of the Products of the Reticulocyte Lysate
System. ................... ‘ ....... 42
Incorporation of Radiolabel into Peptidyl-tRNA ......... 42
Calibration of the Bio-gel A 0.5 m Column . .......... 50
Identification of Homozygous 3112 (Val/Val) Rabbits ...... 60

Assessment of a and Total Globin Synthetic Requirements for
Potassium and Magnesium Ions ............... 61

Labgling of an 2(Val/Val) a Polysomes with
[HL Isoleuc1ne . . . . . . . ....... . ......... 65

Double l:Lzbel Analysis of Nascent Polypeptides Using
C] Isoleucine and L- [3 H]- ~Tryptophan . . . . ...... 74

Characterization of the Bio-gelL P- H3 (-400 mesh) Analytical
System for Quantitationfi [HJ- Tryptophan Labeled

Tryptic Peptideg . . . . ...... . . . . . . . . .75
Quantitation of L-[ H]- Tryptophan-Labeled Tryptic Peptides
by High Pressure Liquid Chromatography ........... 82

Determination of the a and 5 Globin Nascent Peptide Size
Distribution by HPLC of Tryptophan Labeled Tryptic
Peptides ................... . ...... 93

Labeling of Reticulocyte Polysgmes with [35$J-N-formyl
methionyl tRNAfme and L-[ H]-Methionine . . . . . . . . . 103

Perturbation of the Nascent Peptide Size Distribution with
L-D-Methylthreonine .................... 113
Perturbation of the Nascent Polypeptide Size Distribution
with a Complimentary Deoxyribooligonucleotide 118

Analysis of the Nascent Polypeptide Size Distribution as a
Function of Polysomal Size ,,,,,,,,,,,,,,,,,

Inhibition of Globin Translation by Sparsomycin and Gougerotin, 132
Thermal Perturbation of the Nascent Polypeptide Size '

Distribution ....................... 159

The Effect of K+ Concentration on the Nascent Polypeptide
Size Distribution ..................... 168
Discussion . . . . ., ........................ 170

 

References ............................. 196

Run

,4'-‘

I.‘ ,p
‘1'.
h .
I“

‘l!,.

‘5":

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure

Figure

Figure

Figure

Figure

Figure

10

11

12

13

14

15

Index of Figures

CM-Cellulose chromatography of the products of the
reticulocyte cell-free synthesis system. . . . . . . . . .

Bio-gel P-lO column chromatography of peptidyl tRNA
“terial O O O O O I O O O O O O O O O O O O O O O O O O O

Elution of peptidyl tRNA material from DE-52 anion
exchange cellulose . . . . . . . . . . . . . . . . . . . .

Size fractionation of L-[3H]-tryptophan-labeled nascent
polypeptides on the Bio-gel A 0.5 m analytical column. . .

Placement of selected amino acid residues in the a and a
globin pOlypeptide sequence. . . . . . . . . . . . . . . .

Calibration of the Bio-gel A 0.5 m analytical sizing
column with the L-[14CJ-tryptophan-labeled cyanogen
bromide peptides of a and 3 globin . . . . . . . . . . . .

Calibration of the gio-gel A 0.5 m analytical sizing
column with the L-[ HJ-leucine-labeled cyanogen bromide
peptides Of a am 8 QIObin O O O I O O O O O O O O O I O O

CM-cellulose chromatography of L-[3H] isoleucine-labeled
products of a 5112 Val/Val reticulocyte cell-free protein
syntheSiZing sySteflle O O O O O O O O O O O O O O O O O O 0

Dependence of a globin and total globins synthetic rates
on magnesium chloride concentration in the 8112 Val/Val
Lysate O O O O O O O O O O O O O O O O O O O O O O O O O 0

Dependence of the a globin and total globin synthetic
rates on potassium acetate concentration . . . . . . . . .

Nascent peptide size distribution of a globin nascent
pOIypeptides C O O O O O O O O O O O O O O I O O O O O O 0

Comparison of the nascent peptide size distribution of a
and total nascent peptides . . . . . . . . . . . . . . . .

Bio-gel P-2 (-400) chromatography of L-[3H]-tryptophan-
labeled globin tryptic peptides from nascent polypeptide
material C O O O O O O O O O O O O O O O O O O O O O O O O

Bio-gel P-2 (-4DO) chromatography of a globin L-[3H]-
tryptophan-labeled and tryptic peptides. . . . . . . . . .

Bio-gel P-2 (-4OD) chromatography of 3 globin [3H]-
tryptophan-labeled tryptic peptides. . . . . . . . . . . .

.43

.45

.51

.54

.56

.58

.63

.66

.68

.70

.72

.77

.79

.81

 

l
Figur

“SUN

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21a,b

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Figure 29

High pressure liquid chromatography of L-[14C]
tryptophan-labeled a globin tryptic peptides. . . . . . . . .84
High pressure liquid chromatography of L-[14]
tryptophan-labeled B globin tryptic peptides. . . . . . . . .86
Preparative high pressure liquid chroamtography of L-[3H]
tryptophan-labeled globin tryptic peptide for subsequent
paper chromatographic analysis. . . . . . . . . . . . . . . .88
Paper chromatography of L-[3HJ-tryptophan-labeled B

tryptic peptide no. 2 obtained by high pressure liquid
Chromatography. O O O O O O O O O O O O O O O O I I O O O O .90
Paper chromatography of L-[3H]-tryptophan-labeled a tryptic
peptide no. 3 obtained by high pressure liquid chromatography

High pressure liquid chromatographic analysis of nascent
polypeptide derived tryptic peptides from selected positions
of the Bio-gel A 0.5 m nascent peptide size distribution. . .95

L-[3H]-tryptophan-labeling of the 312 and 314 tryptic
peptide as a function of the Bio-gel A 0.5 m distribution
COEffiCi ent O O O O O O O O O O O O O O O O O O O C O O O O .98

L-[3H]-tryptophan-labeled nascent peptide size distribution
used for tryptic peptide analysis . . . . . . . . . . . . . 100

a and e globin nascent peptide size distribution as
determined from HPLC analysis of [3 H] tryptophan- -labeled
tryptic peptides. O O O O O O O O C O O O O O O O O O O O O 102

Time course of [35S]-formylmethionine incorporation
into protein. . . . . . . . . . . . . . . . . . . . . . . . 107

L-[355]formylmethionine/L-[3HJ-tryptophan-labeling of

. the a and B globin nascent polypeptides . . . . . . . . . . 109

L-[3H]methionine-labeling of the a and a globin nascent
polypeptides. . . . . . . . . . . . . . . . . . . . . . . . 112

The effect of L-O-Methylthreonine on the a and total nascent
peptide size distributions. . . . . . . . . . . . . . . . . 115

Subtraction of L-[3HJ-tryptophan/L-[14C]-isoleucine-
labeled nascent peptide size distributions obtained with
and without L-O-methylthreonine treatment . . . . . . . . . 117

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

Figure 35

Figure 36

Figure 37
Figure 38
Figure 39

Figure 40

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

The effect of tetradeoxycytidylate on the tryptophan-
labeled nascent peptide size distribution. . . . . . . . . .

The effect of tetradeoxycytidylate on the a globin nascent
peptide size distribution. . . . . . . . . . . . . . . . . .

The a globin nascent peptide size distribution as a function
Of poI'ysomal Si ze. O O O O O O O O O O O O O O O O O O O O O

The effect of 0.5 x 10"6 M aurine tricarboxylic acid on
the rate of a globin synthesis . . . . . . . . . . . . . . .

The effect of low levels of aurine tricarboxylic acid on the
size distribution of a-nascent peptides. . . . . . . . . . .

Effect of sparsomycin and gougerotin on the rate of globin
synthe51s. O O O O C O O O O 0 O O O O O O I O O O O O O O O

Dosage dependence of the rate of protein synthesis on
sparsomycin concentration. . . . . . . . . . . . . . . . . .

Effect of cycloheximide on the rate of globin synthesis. . .
Effect of cycloheximide on the rate of a globin synthesis. .

Dosage dependence on the rate of total globin and a globin
synthesis on cycloheximide concentration . . . . . . . . . .

Evaluation of the levels of cycloheximide, sparsomycin and
gougerotin which provide 70% inhibition of protein
synth851s. O O O O O O O O O O O O O O O O O O O O O O O O O

The effect of low levels of sparsomycin on the a globin
nascent peptide size distribution. . . . . . . . . . . . . .

The effect of low levels of gougerotin on the a globin
nascent peptide size distribution. . . . . . . . . . . . . .

Sedimentation profile of sparsomycin inhibited, control
and mixed (inhibited and uninhibited) polysomes. . . . . . .

The effect of low levels of sparsomycin on the size
distribution of a nascent peptides from small polysomes.

The effect of low levels of sparsomycin on the size
distribution of a nascent peptides from medium sized
palysomeSo O O ’ I O O O O O O O O O O O O O O O O O O O O O O

The effect of low levels of sparsomycin on the size
distribution of a-nascent peptides from large sized
polysomes. . . . . . . . . . . . . . . . . . . . . . . . . .

121

123

126

129

131

134

136
138
140

142

144

148

150

153

. 155

157

159

Figure 47

Figure 48

Figure 49

Figure 50

Figure 51

Figure 52

Figure 53

The effect of L-[3H]-isoleucine-labeling conducted at
15°C on the a nascent peptide size distribution. . . . . .

The effect of a temperature shift from 37°C to 22°C on
the nascent peptide size distribution. . . . . . . . . . .

The effect of a temperature shift from 37°C to 15°C on
the a nascent peptide size distribution. . . . . . . . . .

The effect of different potassium levels on the a and a
globin nascent peptide size distribution . . . . . . . . .

A comparison of nuclease susceptible regions of a proposed
a globin mRNA structure and the accumulation of nascent
B 910b1n mptideSO O O O O O O O O O O O O O O O O O I O 0

Correlation between proposed regions of single stranded
a globin mRNA structure and the B nascent peptide
aCCWUI ations. O O O O O O O O O O O O O O O O O O O O O 0

Location of nascent peptide accumulation on the secondary
structure of the 5' region of the a and a globin mRNAs . .

162

166

168

171

189

192

194

Table 1

Table 2

Table 3

Table 4

List of Tables

 

Analysgs of Globin Polypeptides for the Incorporation
of L-[ H]-Isoleucine into a and a Globins by $05
Polyacrylamide. . . . . . . . . . . . . . . . . . . . . . . . 62

The Positions of the a Globin Nascent Peptide
Accumulations as Determined by L-Isoleucine Labeling
Of 8112 val/Va] ”sates O O O O O O O O O O O O O O O O O O O 74

The Positions of the a Globin Nascent Peptide Accumulations
as Determined by Tryptic Analysis . . . . . . . . . . . . . .105

The Recovery of [3H]dpm Present in Peptidyl tRNA Relative
to Control dpm ([14C]) From Polysomes Following Thermal
Shift to 22°C and 15°C From 37° C. . . . . . . . . . . . . . .165

 

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Introduction

RNA Primary Structure

Polyribonucleic acids are naturally occurring or synthetic polymers
of nucleoside monOphosphates. These are the monophosphorylated deriva-
tives of B-D-ribofuranosyl adenine, guanine, cytosine and uracil.
Natural polynucleotides contain varying amounts of modified bases. In
polyribonucleotides other than tRNA these modifications are predominantly
the base N-methyl and 2'-0-methyl-ribose derivatives. The sequence of
bases contains not only the information necessary to direct the synthesis
of proteins as in the case of messenger RNA but presumably also contains
information which results in the folding of the polynucleotide backbone
into a unique molecular conformation necessary for the determination of
certain aspects of the functional behavior of the polynucleotides such as
specific initiation rate, and metabolic half life (1,2). These functions
also include interaction of the polyribonucleotide with various specific
proteins (3), specific molecular interactions with regulatory cations (4)
as well as internucleotide interactions which occur at many levels of the
protein biosynthetic apparatus (5). A number of studies to be mentioned
below have indicated that knowledge of the primary structure of a poly-
nucleotide will not immediately describe all of the structural relation-

ships which are needed to account for known RNA function (6).

RNA Secondary Structure
Rich and co-workers (7) published definitive studies of the crystal
Structures of the self-complimentary dinucleoside monophosphates, ApU and

6pc. Analysis of the crystal structures of these molecules permitted

observation of the C-2 rotational axis of symmetry between the two halfs
of the (ApU)2 and (GpC)2 dimers and provided, for the first time,

direct visualization of a segment of a right-handed helix exhibiting
Watson-Crick base pairing. Additional information from these studies
indicated that the sugar phosphate backbone promoted the spontaneous
formation of N-C base pairing in helices of nucleotides (8). These
studies also confirmed predictions of strong base stacking interactions
among the dinucleoside monophosphates in solution. Extension of these
studies to helical polyribonucleotides and polydeoxyribonucleotides
revealed tendencies towards large scale helical ordering.

Helical polynucleotides are described physically by their respective
genus. This term reflects ranges in the axial translation (h) and rota-
tion (t) nucleotide. These parameters may vary from 2.56A-3.14A for (h)
and from 30°-45° for (t).

Genus A helices are characterized by C(3') endofuranose conforma-
tions and a large range of values for (h) (2.56-3.29 A) as well as a
restricted range for (t) exhibiting values from 30-32.7°. This is in
contrast to genus B helices which exhibit a preference for C3' exo- or
C2' endofuranose conformations, narrower ranges of (h) values (3.04-3.4
A) and a broader range of (t) from 31.3° to 45°. The A genus helics have
their bases positioned 3 to 5 A forward of the major helix axis and
tilted positively with respect to a normal with the major axis in con-
trast wfith genus B helics in which the bases are translated to the rear
'of the helix major axis by 0-3 A and exhibit a negative base tilt (9).
These parameters reflect differences in helical dynamics, stability and
configuration which can occur to varying extents in any helical region or

group of helices. In particular, the forward positioning of the bases in

2

the A helix creates a deep groove opposing the glycosidic linkages pro-
viding an opportunity for tertiary interactions such as triple strand
fbrmation as is found in tRNA (10).

Natural RNA helices such as those found in localized regions of mRNA
molecules seem to fall predominantly into the A genus (11). However, it
cannot be excluded that short regions of a particular sequence may result
in unusual and potentially important alternate helical forms. This has
been found to be the case in DNA following the observation by Rich and

coworkers of an extreme conformational variant termed Z-DNA (12).

Optical Properties of RNA Helices

 

One of the best known properties of helical polynucleotides is the
dramatic decrease in the extinction coefficient per residue of the pri-
mary ultraviolet absorption transition upon transfer of a nucleotide
residue from a random coil to a helical environment. This change is
primarily due to the interaction of the aromatic electrons of each base
residue with bases stacked above and below it in the helix. This phenom-
enon is known as base stacking and is reflected in a hypochromic shift
upon helix formation and is thought to be the primary stabilizing force
for helix formation providing a hydrophobic environment for Watson-Crick
hydrogen bonding which in turn leads to the high specificity or compli-
mentarity of the helical interaction. The technique of thermal denatura-
tion and the resultant hyperchromic shift has been used to characterize
the helical content of many natural and synthetic polyribonucleotides.
Using this property, total mRNA, 18S and 285 ribosomal RNA, ovalbumin
mRNA and procollagen mRNAs were found to have from 55 to 75% of their

bases present in base paired fonn (13). It has also been determined that

3

in the case of ovalbumin mRNA (14) and globin mRNA (15) greater than 90%
of the helical structure is preserved at physiological values of ionic
strength, pH and temperature. D'Malley gt a1. were able to carry these
analyses further to demonstrate, with the aid of high resolution thermal
denaturation and heat capacity measurements, that different mRNAs had
characteristic dH/dT profiles which suggested the presence of discreet
helical structures of different classes of stability and of varying
amounts in tRNA, 18S and 28S rRNA, globin, ovalbumin mRNAs and MS-Z RNA.
These studies also indicated the absence of long GC rich structures in
ovalbumin mRNA in contrast to data obtained for tRNA and 28S RNA (14).
In general, it was found that mRNAs had fewer GC rich regions than is the
case for 285 RNA. This result was taken as evidence that the secondary
structure present was not due to random complimentarity of sequence,
which would be expected to give upwards of 50% helicity (16), but rather
was an indication of specific stable structures present in the RNA
molecular structure.

Similar results have been obtained using the techniques of Optical
rotatory dispersion and circular dichroism (15). Globin mRNA showed a
positive Cotton effect which upon shift of the temperature from 20° to
85°, gradually decreased with a concomittant shift of the 285 nm peak and
zero rotation crossover point to longer wavelengths. Decrease in the
Cotton effect is characteristic of loss of asymmetry during the helix
coil transition. This process was fbund to be reversible upon cooling
indicating that renaturation to the original state or to a very similar
state had occurred. The reversibility of nucleic acid denaturation is
well substantiated in the case of tRNA but remains only partially
substantiated by experiment in the case of structural studies on larger

m0l ecul es.

Thermodynamic Properties of RNA Helices

Pioneering work of Uhlenbeck, Tinoco and others on the thermody-
namics of formation of homopolymeric (17) mixed sequence complimentary
(18), and imperfect helices (19), and helical fragments from natural
sources (20) has resulted in the accumulation of a large body of data
concerning the thermodynamics and stability constraints on helix forma-
tion. Evaluation of the enthalpies and entropies for formation of the
dimeric units of helical structures and recognition and characterization
of the contributions of nearest neighbor base pairs, helix nucleation,
and the steric constraints of loops and bulges to overall helix stability
has culminated in a set of parameters which allow systematic estimation
of the free energy of formation of helices of known primary sequence (21)
and provide a systematic criterion by which the relative stabilities of
alternative helical representations of a given sequence may be adduced
and the most stable or probable structure chosen.

These parameters have been coupled with algorithms (22,23) which
allow searches to be made of all possible base pairing schemes for a
given polynucleotide primary structure. These results provide hypothe-
lical polynucleotide secondary structures based on maximal hydrogen
bonding and minimized free energy content. These highly tentative struc-
ture proposals will gradually be supported or rejected as the apprOpriate

experimental approaches become available.

Experimental Approaches to RNA Structural Analysis
It has been pointed out by Fresco and demonstrated experimentally
with synthetic random sequence polynucleotides (16) that a random poly-

nucleotide sequence would be expected to exhibit base pairing involving

50% or more of the bases. It has been argued that the levels of base
pairing found in natural polynucleotides is in many cases only slightly
higher than the amount estimated for a random sequence. This model pre-
dicts that no critical conformation or molecular state need exist for a
particular polynucleotide. Initial experimental evidence at variance
with this position was obtained demonstrating that random polymers had
very low thermal stability reflecting lowered Tm's and shallow hyper-
chromic transitions concomittant with thermal denaturation. This result
was in contrast to natural RNAs which exhibited similar amounts of base
pairing but had distinctly sharper hyperchromic shifts upon denaturation
indicating a high cooperativity of melting as well as significantly
higher Tm values for these transitions. More evidence has accumulated in
support of the view that natural RNAs will have discreet molecular struc-
tures. The early studies of Tinoco gt_al. (24) and Nrede gt £1. (25) on
the ability of tRNA and 5S RNAs to bind some complimentary oligonucleo-
tides to regions of a known polyribonucleotide primary sequence while
other complimentary oligonucleotides would not associate supported a
model of secondary structure composed of helical elements, inaccessible
to the complimentary oligonucleotides probes, separated by accessible
single strand sequences. These techniques allowed demonstration of
single strandedness at the anticodon, amino acid acceptor stem and T U
loops of tRNA.molecules in solution.

Further evidence for discreet molecular conformations in larger
polynucleotides came from the work of Adams gt 31. (26). Using limited
nuclease digestion of R-17 bacteriOphage RNA protected by initiation
complex formation these workers isolated in high yield, and sequenced, a

55 nucleotide long fragment which exhibited extensive helical structure

in solution. The increased sensitivity of single stranded regions of
polynucleotide to nuclease attack relative to double stranded regions
came to the foreground during efforts by Fiers 31; _al_. to determine the
primary sequence of the bacteriOphage MS-2 genomic RNA (28). It was
observed that limited digestion with nucleases such as T1 released
certain fragments in high yield from the polycistronic RNA. Further
analysis showed that different guanylate residues varied dramatically in
their susceptibility to enzymatic cleavage by T1 nuclease. Completion
of the sequence of the MS-2 RNA led to the proposal of an extensively
base paired secondary structural map of the RNA molecule in which the
residues of highest nuclease susceptibility resided predominantly in
single stranded regions while guanylate residues which were hydrolyzed
lew‘ly, if at all, under the same conditions resided in the regions of
proposed stable helical structure (28).

Further exploitation of this approach was used by Flashner and
Voumakis (29) to demonstrate discreet and different patterns of nuclease
resistant fragments characteristic of the rabbit a and a globin mRNAs
indicating the presence of helical elements of structure characteristic
0f the rabbit a and B globin mRNAs. a and B globin mRNAs which had been
raleiodinated were subjected to limited digestion with the single strand
Specific nuclease 51. Analysis of the products by denaturing pol y-
acry] amide gel electrophoresis demonstrated that a limited set of frag-
ments’ which were different for a and B RNAs, had been released under the
(”Millions of the incubation. This result was interpreted as evidence
for unique discreet molecular conformations for the a and B gl‘obi n nRNAs
3'“ indged inconsistent with a model for RNA structure based on a large

"umber of random molecular structures. The susceptibility of specific

7

regions of natural polynucleotides has been noted by others. Hela cell
5.8S ribosomal RNA was observed to have two internal sequences suscepti-
ble to $1 nuclease hydrolysis which allowed the proposal of a hypothe-
‘tical secondary structure consistent with this observation (30). T1
r-ibonuclease digestion of human, hamster and Xenopus laevus 18S ribosomal
IINA also showed limited sensitivity to the nuclease used as a structure
[arobe and led Maden gt al. (31) to propose that the 3' terminus of this
RNA. existed in a highly exposed conformation in the intact ribosomal
subunit. Maden supported the possibility of interaction of the 3'-ter-
mini of 18S ribosomal RNA with other components of the protein synthetic
apparatus.

Nucleolytic enzymes as structural probes have recently been used to

"Remy: regions of secondary structure on the polynucleotide backbone of
Several RNAs. Hurst _eta_l_. (32) have developed a methodology based on
the rapid RNA sequencing techniques Gilbert and Maxam (33).

RNAs which have been terminally labeled with 32Phosphorous are
$41 bjected to limited hydrolysis of $1 nuclease or T1 ribonuclease.
The products are then separated on high resolution denaturing polyacryla-
m‘i de sequencing gels. Bands appear on the gels at positions correspond-
'i 'Dlssy to nuclease susceptable single stranded sites along the polynucleo-
t‘i tie sequence. This method allows determination of the relative suscep-
1tL7i laility and hence accessibility of each phosphodiester bond to the
"‘4 cl ease structural probe throughout the molecule. These data can be
used to support a particular hypothetical secondary structure at the
e)<I:~ense of possible alternative structures.

An alternative approach was used to determine interacting sequences

i '1 large RNA molecules and, in particular, the 16S ribosomal RNA from

8

 

This method uses nucleases to hydrolyze the polynucleotide pre-

E4911-
ferentially at single stranded regions to release helical fragments.

These fragments are separated under non-denaturing conditions which allow

the halves of the helices to remain intact. The separated fragments are

then subjected to electrOphoresis at right angles to the original separa-
tion under denaturing conditions and interacting sequences appears as a
linear array of spots along the direction of the second separation pro-

cedure. The approach provided evidence allowing Brimacombe and Ross to
propose a secondary structure map of the 16S ribosomal RNA and demon-
strated several long range intramolecular interactions (34). These

studies, cited above, are consistent with conserved unique structural
conformations in a given class of RNA molecules and begin to provide a

molecular basis for various functional interactions which have been

observed.

_Sicondary Structure and RNA Function
Early work attempting to relate RNA structure to RNA function began

1" the 1960's. Work by Lodish utilizing f-2 bacteriophage RNA addressed
the Sensitivity of the translational process to severe perturbants of
gee("Hilary structure (35). Using reagents such as formaldehyde to perma-
Denim), denature regions of the phage mRNA it was found that unrestricted
initiation occurred at the correct initiator codon resulting 1" a large
EXCESS of the normal polypeptide products. In addition, .a large nunber
o polypeptides were generated which were found to bear no relationship
to the primary sequence of the normal phage proteins implying that
e"T‘Orleous initiation had occurred at various points other than the true

i ~ .
“‘tiation codon. These results were taken as evidence that secondary

structure which was specifically removed by formaldehyde treatment was

necessary for the correct functioning of the RNA.
In other studies, the coat proteins of the RNA phages f-2, MS-2 and

R-17 were found to play a key role in the regulation of the synthesis of

the product of the RNA polymerase cistron (36). Later studies showed

that the coat protein bound specifically to a helical region of the RNA

. 1

which contained the initiator AUG for the phage polymerase enzyme and

,(Jz...

thus affected its protection from nuclease attack allowing isolation of
the protein binding fragments.

Analysis of the primary and secondary structure of the MS-Z genome
by Fiers _e_tfl. (28) provided a molecular explanation for the effect of
coat protein binding on suppression of replicase production through bind-

ing of coat protein to a helical fragment of the phage RNA which contains

the replicase initiation codon. In addition, the secondary structure of

the coat protein/replicase initiation region, as determined by Fiers 33;
fl- .‘ Provides confirmation of a prOposal by Lodish and Robertson that
part of the coat protein cistron must be translated to expose the viral
Ir‘elfl icase AUG initiator codon since the initiator codon appears to be
hea‘” 15' involved in secondary structure with a region of the coat protein
9e“°me (37). These results led to the proposal of a molecular explana-
tion for the strongly polar effect of coat protein synthesis on replicase
synthesis (28).

Additional evidence for an influence of secondary structure on the
events leading to initiation of protein synthesis was obtained by Pav-
‘akis _a_t 31. (38). These workers used nuclease probes of primary and
Secol'ldary structure, as described above, to show that the 5'-terminal

T‘ .
e910ns of the a and B globin mRNAs of mouse and rabbit possessed ele-

10

ments of structural similarity between the homologous a or B nRNAs

respectively of each species. Analysis of structure revealed that the

initiation codon and flanking sequences in the mouse and rabbit o nRNAs

were inaccessible to nuclease structure probes. This finding was in

contrast to the extensive nuclease susceptibility of the AUG codons in

the homologous a mRNAs. These results may be of considerable functional
significance since it is known that the 8 mRNA initiates synthesis of the

3 globin polypeptide at about 1.5 times the rate of a globin initiation

in the rabbit reticulocyte (1).
Other aspects of the initiation process have been studied within the

Robertson and Legon performed a series of

context of mRNA structure.
These

studies on ribosome binding to radioiodinated globin "RNA (39).
workers were able to isolate and sequence a specific fragment which was

protected from nuclease digestion by bound ribosomal subunits. These

studies revealed that the 405 subunit protected a fragment approximately
30 nucleotides in length from nuclease degradation whereas the complete
305 r‘i bosome protected a much smaller portion of the sane region of RNA
0f approximately 40 nucleotides, suggesting a conformational change in

the DO‘lynucleotide backbone during construction of the preinitiation and

i "1 ‘11 ation complexes.

WGlobin mRNA, Sequence and Translation
The question of the relevance of mRNA secondary structure to mRNA

f .
unction has been evaluated from other points of view. Analysis of the

primary sequence of coding and noncoding regions in terms of the degree

of

Conservation of sequence in homologous RNAs from different species,
codon usage, and the presence of highly conserved oligonucleotide

11

r‘;

sequences, has had considerable impact on the question of the role of
secondary structure in mRNA function.

Early studies of the globin nRNAs from rabbit revealed the presence
of the conserved hexanucleotide, AAUAAA within the 3'-noncoding region of

the a and 5 globin mRNAs (40,41). This same sequence has been found in

the 3' noncoding region of all other pol y(A) containing mRNAs sequenced

thus far.

The function of this hexanucleotide is not known at this time.
The hexanucleotide ACACUU is found to be present within the 5' regions of ‘ “

both a and B globin mRNAs. Although no function is known for this hexa-

nucleotide its conservation would seem significant given that the a and a
5' nontranslated regions bear little other sequence homology. Presumably
there has been an accumulation of a relatively large number of mutations
in this region of the RNA molecules since divergence of the a and 3
sequences.

Sequence analysis of a number of eukaryotic mRNAs has demonstrated

the nonrandom use of synonymous codons. Work by Adams _e_t_ 31. (27) 0"
sequence analysis of the 57 nucleotide long region of the coat protein
(fist-"On of R-17 bacteriophage indicates that degeneracy in the genetic
code and nonrandom usage of synonymous codons has allowed maximization of
sectNiclary structure without alteration of the resultant polypeptide
Seq‘Jence. This view has been carried further by Ball who suggests that
some of the selective pressure on an amino acid sequence reflects changes
which maximize secondary structure (42% This proposal was suggested
f0} 1owing sequence analysis of the MS-Z coat protein gene. It was demon-
strated that the codons for the most conserved amino acids occur in

extensively base paired regions. Although it is difficult to make con-

Q‘ Usive statements from such studies, sequence analysis and asymetric

12

‘

codon usage argue in favor of structural constraints on certain aspects

of mRNA function.

_mfiNA Translational_Dynamics
The globin biosynthetic system has been extensively studied in terms

of the rate and dynamics of the process of protein synthesis. Theore-.

ti cal studies by Gibbs 33:31. on template directed polymerization

processes allowed prediction of some of the properties found in protein

synthetic systems (43,44). These workers indicated, as Lodish later

underscored (45), that for polymerizing systems for which the initiation

step is rate limiting, inhibition of initiation will result in more

severe restriction of a slower initiating messenger RNA relative to a

faster initiating nRNA. Later studies using inhibitors of mRNA initia—

tion allowed demonstration of this phenomenon with the a and a globin
mRNA- Jacobsen gal. (46), building on previous studies which estab-
lished that the overall (average) rate of elongation and termination were
mUQh'ly equal for a and a globin nRNAs, determined that the a globin "RNA
in-i ti ates synthesis only 60% as efficiently as the 8 mRNA and that there
is approximately 1.4 times as much a globin nRNA as a "RNA resulting 1n a
“Qt ratio of a to a synthesis of unity. Although there were technical
di ff’lculties with both of these studies, the conclusions of Jacobsen and
Lodish concerning the differences in apparent initiation rates and the

6331 gnment of rate limiting step in globin synthesis at the level of

1n‘ti at1on remain valid.
Other studies involving analysis of the effects of elongation inh1-

bitfirs such as gougerotin, cycloheximide and amicetin and the initiat1on

‘nhibitors edeine and pactamycin established the dependence of polysome

13

size on the initiation and elongation rates. Evidence was obtained that

the number of ribosomes on a mRNA at a steady state is proportional to

the length of the coding region of a nRNA and to the rate of initiation

of protein synthesis. The mean polysomal size is inversely dependent on

the rate of elongation and termination.
of mRNA structural and functional half life seem to indicate that control

Recent studies on the regulation

of nRNA ribosomal loading may be used to effect control on nRNA turnover
Cohn $.11: used elongation and initiation inhibitors to demon-

(47).
st rate that certain of the T7 early mRNAs are significantly protected

from nuclease inactivation when they are present in larger polysomes.
This result was taken to indicate that ribosomes may be spending more of

their time residing over nuclease susceptable sites in the mRNA, reducing
Other

the effective level of that site as a target for nuclease attack.
mRNAs were found to be stabilized by inhibitors which allowed ribosomal

density to increase near the initiation region, indicating the possibil-

ity of ribosomal protection of a nuclease susceptible site near the

initiator region. These studies raise the question of whether structural

a'-"pe<:1;s of a mRNA can modulate pol ysomal size through effects upon the

r‘ates of initiation, elongation and termination and in turn upon ribo-

some“ density on a mRNA thus altering the observed half life.
A fairly large body of evidence is accumulating concerning the

effefit of mRNA secondary structure on the rates of elongation and termin-

at‘i On. Protzel gal. have demonstrated that the size distribution of

t .
he nascent polypeptide intermediates of globin synthesis is nonuniform

(48). Gel chromatographic analysis of uniformly labeled nascent polypep-

‘des conducted under denaturing conditions has been used to demonstrate

14

‘ua

.2513.- ‘—

F
_ AIL-

r

that the population of nascent polypeptide intermediates is composed
predominantly of discreet size classes of nascent polypeptides which are

disproportionately represented throughout the p0pulation of a and B

globin nascent polypeptides. Since the nascent polypeptide molecular

weight uniquely corresponds to the position of the associated ribosome

along the coding region of the particular mRNA being translated, the

disproportionate representation of a particular size class of nascent

polypeptides was taken as an indication of an increased probability of

finding ribosomes occupying this region of the mRNA. The most likely

explanation for this observation is that the rate of ribosomal

translocation is not uniform but changes locally along the mRNA. Later

work by Chaney and Morris sought to identify the origin of the

nonuniformity of local translocation rates (49). These workers were

unable to identify any rate limiting components of the protein

bios.Ynthetic apparatus which could account for the production of

nonuniform elongation rates during globin synthesis. Translation of

PO].Y(A)+ RNA from rabbit reticulocytes in a wheat germ cell-free protein

synthesizing system permitted demonstration of an identical nascent
pePt‘i de size distribution to that observed in the reticulocyte system.
ThesE findings were taken as evidence that the origin of local nonuniform
rates of elongation was associated with some property of the nRNA itself
and not due to a limitation of some component of the biosynthetic

appa Y‘atus.
Further studies sought to analyze the nascent peptide size distribu-

1:10“ in a different system reflecting the translation of a single mRNA

Seqtlence. Chaney et al. observed a nonuniform size distribution of nas-

Cent polypeptide precursors in the synthesis of MS-Z coat protein (50).
The nascent peptide accumulations were used to map points of slow

15

translocation on the coding region of the coat protein cistron and these
results were compared with the "flower" conformation pr0posed by Fiers gt
1L. as the most stable secondary structure for this part of the MS-2 RNA

molecule. It was found that the accumulations of nascent peptides corre-

sponded to single stranded stretches at or near the 5' side of regions of

stable helical structure. This result was taken as evidence that helical

regions of the RNA molecule retard ribosomes entering these helical

regions. This model predicts that ribosomes will tend to occupy regions
of the mRNA at the points of entry into regions of secondary structure.
This model provides a basis for the formulation of a mechanism by which

the amount of stable helical structure in a mRNA may modulate the average

elongation rate by virtue of its effect on local translocation rates

which contribute to the average elongation rate. Independent evidence

for this is indicated by the observation of Schimke _e_t _a_1_. (51) that
irreversible denaturation of several nRNAs by treatment with methyl
mercury hydroxide resulted in an increase in the rate of elongation as
"91 1 as an increase in the ability of these mRNAs to direct the synthesis
0": full length reverse transcripts instead of partial transcripts.
Part1 a'l reverse transcripts are thought to arise from premature termina-
tion brought about by stable structures present in the template. These
a"‘Qufllents indicate that mRNA secondary structure may be involved in a
dua] role in determination of mRNA half life, directly through modulation
of the amount of nuclease susceptable regions per mRNA molecule and
i "directly by providing a mechanism by which\ ribosomal coverage of
s‘usttteptable sites along the polynucleotide backbone may be maximized.
The goal of this thesis is to determine which nascent peptide

at:Cumulations, previously observed in the synthesis of the rabbit a and

16

1..er

B globins, correspond to a specific and 8 specific nascent peptide
accumulations, to correlate the single mRNA specific nascent peptide
accumulations thus obtained with any physical data on the a or 3 globin
mRNAs which may become available during the time this work was in
progress and finally, to test the ability of perturbative techniques
applied to the protein biosynthetic apparatus, eg. inhibitors,
temperatures, etc. to provide information relevant to the hypothesis
analyzed herein, that mRNA secondary structure directly hinders ribosomal

translocation along regions of the mRNA involved in stable secondary

structural interactions.

17

Materials

Cycloheximide, guanidinium chloride (practical grade), bovine hemin

(twice recrystallized), phosphocreatine, 2,4-dinitrophenyl alanine,
Triton X-114, N,N,N',N'-tetramethylethylene-diamine, and creatine phos-

phokinase (E.C. No. 2.7.3.2) and aurine tricarboxylic acid were purchased

from Sigma Chemical Co., St. Louis, Missouri. Phenylhydrazine-HCI, . .
napthalene (scintillation grade), and diethylpyrocarbonate were purchased . [F

from Aldrich Chemical Co., Milwaukee, Wis. Gougerotin was purchased from
Calbiochem Co., La Jolla, Calif. Bio-Rad Co., Richmond, Calif., was the

source of Bio-gel A 0.5m (10% agarose) and Bio-gel P-10.

(sodium pentobarbital) was obtained from Abbott Laboratories, North

Nembutal

Chicago, Ill. Research Products International was the source of 2,5-di-

phenyloxazole (PPO, scintillation grade), 1,4-bis 2-(4-methyl-5-phenyl-
oxazolyl)-benzene (POPOP, scintillation grade), and Triton X-100. Blue
dextran 2000 was purchased from Pharmacia Co., Uppsala, Sweden. Micro-
granular (preswollen) diethylaminoethyl cellulose (DE52) and carboxy-

methyl cellulose (CM32) were obtained from Nhatman Biochemicals, Ltd.,
Maidstone, Kent, Great Britain, as were GF/C glass fiber filters. Sodium
heparin was purchased from Fisher Scientific Co., Fair Lawn, New Jersey.
Spa rsomycin was generously donated by Drug Research and Development,

of Vision of Cancer Treatment, National Cancer Institute, Bethesda, Md.

Radioactive amino acids were purchased from New England Nuclear,

BOSton, Mass. ([3HJ-tryptophan. 7-9 Ci/mmole, [14CJ-tryptophan,
50- 7 mCi/mmole, [3HJ-isoleucine and [14CJ-isoleucine) and Amer-

18

 

sham/Searle Co., Arlington Heights, Ill. ([3HJ-leucine, 60 Ci/mmole.
Ultra pure grade sucrose was purchased from Bethesda Research Labs,
Bethesda, Md.

All other chemicals used were reagent grade.

19

Methods

Preparation of Rabbit Reticulocytes
New Zealand white male rabbits weighing 6-10 lbs were made anemic

with four daily injections of 2.5% phenylhydrazine in an isotonic sodium

potassium and magnesium salts solution at pH 7.30 as described by Allen

and Schweet (52). Following a recovery period of three days rabbits were

i nj ected with a solution composed of 100 mg sodium nembutal and 2000
units of sodium heparin in a final volume of three ml via the marginal

Exsanguination was accomplished by cardiac puncture with a 100

ea r vein.
Blood so obtained was

m1 glass syringe fitted with an 18 gauge needle.
i nmediately chilled to 0°C on wet ice and all subsequent operations were

performed at 0-4°C. Hematocrits from such preparations ranged from

9‘15%
Blood was initially passed through two layers of glass wool to

e"”“Fect removal of small pieces of tissue and then centrifuged at 4000 x g

x 20 minutes in a Sorvall GSA type rotor. Plasma was removed by aspira-

ti on and considerable care was taken to remove the buffy coat from the
top of the pellet. Cells were resuspended with a rubber policeman in a
V0] ume of Ringers saline (RS) solution approximately equal to the plasma
V01 ume (53). Cells were then isolated by centrifugation at 4000 xg x 10
mi rIutes followed by aspiration of the RS supernatant. This washing

p"‘0<:edure was repeated three times to give cells which had been washed

and pelleted four times.

20

Preparation of the Reticulocyte Lysate

Washed rabbit reticulocytes were lysed with an equal volume of ice
cold sterile water. The lysate was then centrifuged at 25,000 xg x 20
minutes. The supernatant fraction was decanted to another centrifuge
tube to ensure homogeneity of the lysate. Aliquots of 700 to 900 pl were
inmediately frozen by injection into vials which had been maintained at
-40°C on dry ice. All glass which the lysate was to encounter was washed
in a solution of 1 N KOH, 50% ETOH, rinsed first with water and then with
a di ethyl pyrocarbonate containing solution and placed in an oven to dry
at 120°C for several days or at high temperature (270°C) for at least 6
hours prior to use. Lysate aliquots were capped and stored in liquid

nitrogen until used.

wtions for Cell-Free Protein Synthesis

The reticulocyte cell-free protein synthesizing system contained in
a total volume of 1 ml: 800 pl lysate, 0.3 mol es of hemin prepared as
described by Darnbrough et al. (54), 11.0 males of creatine phosphate as
the sodium salt, 100 ug creatine phosphokinase at a specific activity of
50 units/mg protein, 1.0 umoles adenosine triphosphate, sodium salt, 0.20
pmo] es guanosine triphosphate, potassium salt, and 0.1 times the final
am? no acid concentrations of Lingrel and Borsook (53).

Potassium and magnesium requirements were determined for each lysate
I3"‘el3aration on the basis of those levels of salt required for optimal
initial velocity and extent of incorporation of L-[3H]-leucine (10 uCi
per 250 pl lysate) into alkali stable 10% trichloroacetic acid precipi-
table material. These Optima were consistently found to be in the range
of 1.2-1.6 millimolar for MgClz and 130-140 millimolar for potassium

acetate.

21

A
f}!

lp-‘o
1

Cell-Free Globin Biosynthesis, Confirmation of Product Identity:

Separation of a and 8 Globin Chains

Analysis of products synthesized by the cell-free system was accom-
plished by CM-cellulose ion exchange chromatography according to a modif-
ication of the method of Dintzis £511. (55). Small aliquots of postri-
bosomal supernatant obtained from the preparative ultracentrifugation of
[3HJ-Leu labeled lysate at 105,000 xg for 180 minutes were dialyzed
against distilled water for 24 hours to remove radioactive amino acids.
The retention was used to prepare globin by the method of Ninterhalter
and Huehns (56). Approximately 2 ml of the hemoglobin solution was
dripped into a rapidly stirring solution of 200 ml acetone and HCl (final
concentration, 0.6 M) maintained at -40°C in a dry ice/acetone bath.
After 30 minutes stirring the precipitated globin was collected by
CEHtrifugation at 13,200 xg x 20 minutes. The pellet was washed gently
"1' th fresh acetone, dried and dissolved in .02 M pyridine, 0.2 M formic
acid. .005 M 2-merceptoethanol (referred to as 1X Dintzis buffer). This
50] ution was cleared of insoluble material by centrifugation at 25,000 xg
for 20 minutes. 35,ooo-so,ooo cpm of the globin solution was applied to
a 0.9 x 22 cm column of CM-cellulose-32 equilibrated with 1X Dintzis
bu“Fer. Following a wash cycle of 10 volumes of Dintzis 1X buffer,
e] “tion was affected with a gradient of pyridini um formate as described
by plr‘otzel and Morris (48). Fractions were collected directly into

1 “mm scintillation counting vials and counted in a xylene based

3‘: l ntillation cocktail.

p
‘rllgflggration of the Peptidyl tRNA - Polysomal Labeling
Labeling of peptidyl tRNA was accomplished in reaction volumes 0.5

22

 

to 1.0 ml containing 50 to 200 uCi of tritium labeled amino acid or 5 to

20 uCl [14CJ-1abe1ed amino acid. when the labeled amino acid was

other than isoleucine or tryptophan, the input level of the corresponding

cold amino acid in the Borsook mix (53) was reduced 10 fold. Incubations

were conducted at 37°C for 10 minutes unless otherwise indicated.
Label ing was terminated by rapid addition of 2 ml of ice cold Medium B
(48) containing 0.21 nM sparsomycin and 0.059 mM cycloheximide.

Polyri bosomes were isolated by centrifugation at 105,000 xg for 2 hours

“'“r
1!

at 2-4OCc

[Association of Polysomal Structures
The polysomal pellet isolated by ultracentrifugation was resuspended
in 0-75 ml of a solution composed of 0.25 M sucrose, 0.059 _nfl cyclohexi-

mide and 0.21 mM sparsomycin. This suspension was then brought to con-

centrations of 3.0 M LiCl, 4.0 M Urea, 0.05 M 2-mercaptoethanol and 0.05

34. NaAc pH 5.6. This mixture was incubated for 16 hours at 0°C followed

by Centrifugation at 10,000 xg x 20 minutes. The supernatant solution

was used for subsequent purification steps.

Pu\""ifjcation of Peptidyl-tRNA
Stock urea solutions were prepared by stirring a 9.0 M solution of

Urea for four hours with Amberlite MB-3. The amberlite was removed by

fi ] tration through a medium grade scintered glass funnel and the volume
adjusted to give a final urea concentration of 8.54 M. This stock urea
so] Ution was used to prepare buffers for subsequent peptidyl tRNA purifi-
cation steps as described by Slabaugh and Morris (57) as modified by
Chaney and Morris (49). Buffer I containing 7.6 M urea, 0.05 M 2-mercap-

23

‘nl

a
bvh

an

(_>

\

(,1
‘—

 

s:

._{7/

toethanol, 0.1 M sodium acetate (pH 5.6). Buffer 11 contained all com-
ponents at identical concentrations as in to buffer I except that the
sodium acetate concentration was raised to 0.75 M.

DEAE cellulose (Nhatman DE-52 in microgranilar preswollen form) was
suspended in approximately 60 ml of 0.5 M acetic acid per 7 grams of ion
exchange cellulose. The fine particulate material was removed by allow-
ing the DEAE cellulose to settle to one third the column height of the m
suspension followed by removal of the volume above the bulk of the set- I “
tling cellulose. This procedure was repeated twice, the volume restored by
to its original value and the pH adjusted to 5.6 with saturated NaOH.

The ion exchange cellulose was then isolated in a scintered glass funnel
and washed three times with Buffer I in amount equal to the original
suspension volume. The cellulose was then resuspended in Buffer I, the
fl he particulate material was removed once and the final volume of the
suspension was brought to twice the volume of the fully settled
Cellulose. Prior to adsorption of peptidyl tRNA a column containing
approximately 2 g of DE-52 cellulose was washed with approximately 50 ml
of Buffer I.

Lithium chloride and contaminating amino acids were removed from the
'“i bosomal dissociation mixture by chromatography on a 1.9 x 40 cm column
of Bio-gel P-10 (200-400 mesh) equilibrated with Buffer I. The column
was eluted with buffer 1 and radioactivity which eluted in the void
V01 ume was pooled and adsorbed to a DE-52 cellulose anion exchange column
and washed with approxaimately 150 column volumes of buffer 1.

After peptidyl tRNA had been adsorbed to the anion exchange material
and washed as described above, the peptidyl tRNA was eluted stepwise by

e] Ution of the column with Buffer II.

24

Fractions containing the radioactivity which was eluted were concen-
trated by pressure ultrafiltration with N2 gas to a final volume of
0.3-0.5 ml in an Amicon model 8 MC microultrafiltration system (Amicon
Corp., Lexington Ma.) over an Amicon UM-2 membrane. Three ml of 6M
guanidinium chloride, 100 _rn_M 2-mercaptoethanol pH 6.5 were then added and
the solution concentrated to 0.3-0.5 ml.

The pH of the concentrated peptidyl tRNA solution was then adjusted
to a value of 13-14 with 6 N NaOH as determined by pH paper and allowed
to incubate for 4 hours at 37°C to cleave all aminoacyl and peptidyl-tRNA
ester bonds. The solution was then adjusted to pH 5.6-6.0 with glacial

acetic acid as judged with pH indicator paper.

mstallization of Guanidinium Chloride

Practical grade guanidinium chloride was recrystallized twice by a
m(Ddification of the method of Nozaki and Tanford (58). 1000 g of guani-
diniun chloride was dissolved in sufficient absolute ethanol at 70°C to
91 Va a final volume of 3500 ml. To this solution was added 1 gram of
activated charcoal followed 5 minutes later with 1 gram of Celite and
the solution filtered while hot through a Whatman 1 filter paper in an
18.5 cm buchner funnel under mild vacuum. Following redissolution of any
<:".ystallized material in the filtrate, 120—200 ml of benzene were added
to the ethanol until crystals first appeared and remained. This solution
was allowed to cool to room temperature and then allowed to stand over-
"1' ght at 4°C. ‘

Collection of the crystals was accomplished by vacuum filtration in
an 18.5 cm buchner funnel. These crystals were then washed with a small

amount of ethanol maintained at -20°C, collected and dissolved in a mini-

25

rIl‘

17"“

*5.

mum amount of absolute methanol maintained at 68-70°C. Once all the

crystals were in solution the solution was allowed to stand at -20°C

overnight. The crystals which formed were harvested as described above

and dried to constant mass over potassium hydroxide pellets under reduced
pressure.

Analysis of Peptide Size Distribution

The size distribution of nascent polypeptides was analyzed by the

981 permeation chromatographic method described by Fish et al. (59) and

by Protzel and Morris (48). Radiolabeled nascent polypeptides derived

1:l"0rn peptidyl-tRNA (above) were subjected to gel permeation chromatog-
ra phy under conditions known to render polypeptide chains in a random

CO‘i] configuration (6.0 M guanidinium chloride, 0.1 M 2-mercaptoethanol,
pH 6.5) on Bio-gel A 0.5m agarose.

Approximately 200 ml of the Bio-gel A 0.5m slurry were brought to a
fi nal volume of 1000 ml and the slurry was allowed to settle to 50% of

the column height of the vessel and particulate material in the upper 50%

of the solution was removed by aspiration. This Operation was repeated 5

ti mes or until no particulate material could be seen above the slurry at
50% column height. The slurry was then allowed to settle completely, the
s“pernatant decanted and the slurry gently resuspended in a guanidinium

chloride solution (at 4°C) equal to twice the packed slurry volume. Once
the slurry had settled this procedure was repeated twice or until the

de"sity of the supernatant was approximately equal to that of the fresh

so1ution at 4°C. A final addition of 6 M guanidinium chloride solution

“as added to give a final volume of 400 ml and this suspension was used

t0 prepare the analytical gel column.

26

 

The slurry was slowly and continuously poured into a Pharmacia
analytical column 100 cm x 1.6 cm fitted with a 500 ml reservoir and

containing 15 ml Of 6 M guanidinium chloride solution. The slurry was

sti rred to the bottom of the column to remove any suspension inhomogen-

eity and, following a 5 minute standing period, the column was allowed to

fl ow under a hydrostatic head of 8 to 10 cm. The outlet was gradually

l owered to approximate a constant flow .rate until the final Operating

hydrostatic head of 65-70 cm was reached. The slurry was allowed to

Continue packing until a bed height of 95 cm had been obtained. The

C01 umn was washed with 3 column volumes of 6 M guanidinum chloride 100 mM

Z—mercaptoethanol solution, pH 6.5. Prior to analysis of any sample, the

C01 umn was allowed to run for at least 24 hr.

The sample prepared for analysis as described above had a final

V01 ume of 0.5 and 0.7 ml. The sample was made 50 mM in dithiothreitol

and sucrose was added to a final concentration of 90 mg/ml. After two

hoHrs at room temperature, 50 ul Of 3.6% Blue dextran and 50 pl of 0.1%

DNP—alanine were added prior to chromatographic analysis to serve as

e”(<21 uded and included volume markers, respectively. The sample was

applied at the top of the column under a column of the running buffer by

1 ayering the sample onto the bed with a pasteur pipet. The column was

developed in the descending direction and 0.9 ml fractions were collected

d1 Pectly into plastic or glass liquid scintillation vials for

determination of radioactivity.

P\eparation of Cyanogn Bromide Fragments of Labeled a and 3 Globin

A check of the calibration properties of the Bio-gel A 0.5 m column

as determined by Protzel and Morris (48) was conducted using the 4

27

 

cyanogen bromide fragments of a and B globin. L-[14CJ-tryptophan

l abeled total (a and B) globins were used as a source of the N-terminal o
and B cyanogen bromide fragments and L-[3H]-leucine labeled globin

provided a source of all 4 a and 3 CNBr fragments. Globin was dissolved

i n 70% formic acid at a concentration of 5.0 mg/ml. A 400 fold molar

excess Of cyanogen bromide was added and the solution was placed in the

dark for 36 hours at 25°C. Following the incubation period, 15 times the

Original sample volume of water was added and the samples frozen and
1 .YOphilized.

 

 

Determination of Distribution Coefficients

Elution data from the size distribution experiments, as determined
by Bio-gel A 0.5 m chromatography in 6.0 M guanidinium chloride, were
tr‘eated as described by Fish et al. (59). The distribution coefficient
(Kd) was calculated according to the formula:

Kd = (Ve-VO)/(Vi-Vo)

Where Ve is the mass of solvent which corresponds to the peak concentra-

tion of the eluting solute.

V0 is the void volume in mass Of solvent.

Vi is the mass of solvent contained within the gel matrix and

C01 umn. In this work these parameters were determined in terms of volume

lnstead of mass.

Blue Dextran 2000 was used as a marker for the void volume and

CH nitrOphenylalanine was used to determine the total volume of the column

and matrix which was accessible to solvent. In the experiments described

herein the location of the blue dextran peak maximum was determined
elther visually or spectrophotometrically at a wavelength of 637.5 nm in
the visible range. The fractions corresponding to the elution maxima of

28

the DNP alanine were determined spectrophotometrically at a wavelength of

360 nm in the near ultraviolet.

Identification of 3112 (Val/Val) Homozygous Rabbits

Identification of individual rabbits homozygous for the substitution
of isoleucine for valine at position 112 of the 8 globin chain present in
the population of white New Zealand male rabbits was determined on the
basis of the absence of incorporation of radiolabel (presented as

L—[3HJ-isoleucine) into purified 8 globin, with incorporation into a

 

91 obin polypeptides as a reference.

White New Zealand male rabbit (8-10 pounds) were made mildly anemic

With a single injection Of 1.5 ml of 2.5% phenyl hydrazine prepared as

described above. After a four day period approximately 5-10 ml of blood

were collected from each rabbit from the marginal ear vein into nalgene
Centrifuge tubes containing 200-500 units Of heparin sulfate per tube.
Subsequent Operations were performed at 0-4°C. After the blood was

Collected and chilled the tubes were balanced and the cells isolated by

Centrifugation at 4000 xg for 10 minutes. The cells were then washed

t\nlice by resuspension in Ringer's saline (60) followed by centrifugation
at 4000 xg for 10 minutes. Washed, packed cells (1 ml) were then trans-
1“erred to glass culture tubes containing per tube 5.0 uCi of L-[3HJ-ile
at a specific activity of 16 Curies/mmole, 0.15 ml of a solution of 10.5

mlel ferrous amnonium sulfate in Ringer's saline and 2.85 ml of incuba-

tion mixture components. The 8112 Val/Val incubation mixture con-

tained, per 100 ml total volume, 13.9 ml of 10X amino acid mixture pre-
Pared as described by Lingrel and Borsook (53) except that isoleucine was
maintained at one tenth the specified final concentration, 30.4 ml

Ringer‘s isotonic saline solution, 1.81 ml Of 0.25 M MgClz, 10%

29

gl ucose, 18 ml 0.164 M Tri5°HCl (pH 7.75), 14.37 ml 1 mM sodium citrate

i n Ringer's saline (1 mM) and 21.56 ml sodium bicarbonate in Ringer's

saline. Cells were su5pended in the labeling medium by gentle swirling

and allowed to incubate for 60 minutes at 37°C. Following incubation

cells were chilled on wet ice and transferred to plastic centrifuge

tubes. Ice cold RS (15 ml) was added and the cells were collected by

centrifugation at 4000 xg for 15 minutes. Following removal Of the

supernatant a lysate was prepared by the addition of 4 ml Of water, 1.0

mM in glutathione. Cells were incubated on ice with frequent swirling

for 10 minutes, followed by removal of cell debris by centrifugation at

17,500 xg for 20 minutes. This supernatant solution was stored at -20°C

and subsequently used to prepare globin as described above.

Mid Estimation of L-Isoleucine Incorporation by SDS-Gel Electrophoresis

The SDS gel electrophoresis procedure used here is essentially that

described by Wood and Schaeffer (61). Hemoglobin solutions prepared as

above were made 0.5 to 1 mg/ml in protein. Samples were made 1% in SDS

and 2-mercaptoethanol and then heated to 100°C for 3 minutes. The mix-

tures were dialyzed at room temperature against 0.1 M sodium phosphate

buffer, pH 7.2, containing 1% SDS and 0.1% 2-mercaptoethanol. Inmediate-

1y prior to application of samples to gels a volume of 0.05% bromophenol
blue dye in 50% glycerol was added equal to 5% Of the sample volume.

The electrophoresis buffer was 0.1 M sodium phosphate, pH 7.2, 0.1% in

SDS. Gels were, composed of 12.5% acrylamide with a bis acrylamide to

acrylamide ratio of 1 to 20. The Bis acrylamide solution was prepared

prior to use by dissolving 6.072 g of acrylamide and 0.304 9 bis acryla-
mide in the electrophoresis buffer. Following removal of particulate

30

1

.411

material by filtration through Whatman 1 filter paper the solution was

degassed and TEMED and ammonium persulfate were added to a final concen—

tration Of 0.05% each. Disk gel columns were poured in 0.6 x 15 cm

si l anized glass tubes to a height of 13 cm and allowed to polymerize at

room temperature. Pre-electrophoresis of the gels was conducted at room

temperature at a current of 2 mA per gel for one hour.

Samples were applied to the gel column in a volume 25-50 ul depend-

i ng on the number of counts per minutes per microliter available. For

these analyses 8,000-10,000 cpm were present in a sample volume of 25 ul.
El ectrophoresis was conducted at 8 mA per gel for approximately 13 hr or

until the marker dye had migrated 12 cm. Cells were stained with cyanin

br‘i lliant G stain (62) for 4-8 hr and destained with 0.02% sulfuric acid

in water. In this system, the band which migrates farthest toward the

anOde is a globin as expected for a separation according to molecular

Wei ght. For this analysis bands were excised by cutting the gel halfway

t”elf-ween the two stained bands and taking gel to a length of 1 cm in both

Cl‘if‘ections. Each gel slice which contained either an a or a 8 band was

then diced with a razor blade, placed in 0.5 ml 30% H202 and incu-

bated at 40°C for 4 hours. Ten ml Of formula C scintillation coctail

Was then added and the samples counted for radioactivity in a Beckman

LS~230 liquid scintillation spectrometer. Formula C scintillation coc-

ta‘il contained in 1 liter, 60.6 g napthalene, 6.06 g 2,5-diphenyloxazole,

0.49 g 1,4-bis[2-(4-methyl-5-phenyloxazolyl)]benzene, 606 ml xylenes and

333 ml Triton X-114. Samples which gave and to 8 cpm ratio of 9:1 or

g"eater were subjected to further analysis by CM-cellose column chroma-

toSJraphy as described above for confirmation of the 8112 val /val pheno-

type. Rabbits determined to be of this phenotype were used to prepare

reticulocyte lysate cell free protein synthesizing system as described
above.

31

 

Preparation of Uniformly Labeled L-LMCLtg) Globins

1.0 ml of the 8112 Val/Val lysate was incubated with the master mix

components as described above and 50 uCi L-[14CJ-tryptophan for one

hour at 37°C. Following exhaustive dialysis against distilled deionized

water, globin was prepared by the dry ice acetone method of Winterhalter

and Huehns (56). Precipitated globin was collected by centrifugation as

described above, dissolved in Dintzis 1X buffer (see separation Of a. and
B globin chains) and chromatographed on a 2.5 x 90 cm coluim Of Bio-gel

P—GO equilibrated in Dintzis 1X buffer to remove a high molecular weight

contaminant. The fractions corresponding to the major component Of

radioactivity (80% of the total cpm eluted) was collected and lyophi-

‘l ized. The globin was redissolved in distilled deionized water, centri-

fllged at 25,000 xg for 20 minutes and lyophilized. Analysis of this

sample by Bio-gel A 0.5 M chromatography in 6 M guanidine hydrochloride,
100 mM 2-mercaptoethanol revealed a single component of average molecular

Wei ght corresponding to that expected for the globins.

hyptic Peptide Analysis Of L-L3H1—Trp-Labeled Nascent Polypeptides

20 ml Of lysate and master mix was incubated with 5 mCi Of trypto-

phan at an initial specific acitivity of 23 Ci/mnole. Purified nascent

DOlypeptides derived from polysomal peptidyl tRNA were fractionated as
described above by Bio-gel A 0.5 m column chromatography except that
every third fraction was counted for radioactivity and the remaining

1tr‘c‘tctions were subjected to tryptic peptide analysis.

Mptic Digestion of Nascent Polypeptides

To each Bio gel A 0.5 m fraction of approximately 400 pl was added
20 ul of a solution containing 1-2 mg/ml uniformly labeled L-[14C]__

32

 

trp globins as an internal standard providing radioactivity, as [14C]

cpm, to an upper limit of 10% of the [311] cpm in each sample. To this

sol ution was added sufficient carrier globin chains to bring the total

91 obin mass to 5 mg. This solution was brought to a final volume of 5 ml

fol l owing the addition Of 250 pl of 1 M CaClz and 1 M TriS°HCl (pH

8- 3) to give a final concentration of 50 _nM CaClz and 50 mM Tris'HCl

(pH approximately 8.0) and 1 mg/ml globin. After equilibration of the

sample under vacuum at 37°C for 10 minutes, 10 ml of a 4 mg/ml solution
01: l'PCK—trypsin (Worthington) was added to give an initial globin to

t-"‘.ypsin mass ratio of 125:1 and allowed to incubate for two hours. Two

additional 10 ul additions were made and each addition was followed by a

2 hr incubation period for a total incubation time of 6 hr. The reaction

was terminated by the addition of 250 pl of glacial acetic acid. Samples

were stored at -20°C until needed for further analysis. Under these

diQestion conditions, no protein precipitate was apparent.

@[aaration of Bio-gel P-2 (—400 mesh) Column Chromatography Buffer

5 moles (300.3 g) of urea were dissolved in sufficient deionized
d1 stilled water to a volume Of 900 ml or 90% of the total final volume.
Following equilibration Of the solution to roan temperature, 25 g of
Amberlite MB-3 mixed bed ion exchanger was added to effect deionization

a"d the mixture was stirred for 4 hr or until the solution had a conduc-

tl' vity less than 1.5 MM at 23°C. The resin was removed by filtration

thr‘Ough a medium grade fitted glass funnel. The solution was then made 5

"'M in 2-mercaptoethanol and sufficient 1 M Tris HCl (pH 8.0) was added to

9‘1 ve a final conductivity of 115-120 uMhO. The solution was then brought

to a final volume of 1 l, filtered through 0.45 p millipore membrane

33

 

(pore size) and stored at 4°C prior to use. Large batches may be made

and frozen at -20°C prior to use. This buffer should not be used if the

conductivity exceeds 200 mm. Each lot Of Bio-Gel P-2 (-400 mesh) must

be tested for the Optimal buffer conductivity for peptide separation.

Preparation of Bio-Gel P-2 (-400 Mesh) Analytical Column

Approximately 85 g Of Bio-Gel P-2 (-400 mesh) column support mater-
ial is added to 200 ml of column equilibration buffer with slow stirring

until all of the support material is suspended. The suspension is then

al 1 owed to stand for 48 hours at 4°C. The support material is then

resuspended and fine particulate material was removed following settling

of the bulk Of the support through 50% Of the total column volume. This
pr‘Oeedure is repeated once and in most cases further removal of fine

pa"‘ticulate material is unnecessary. The support material is then

r‘esuspended in a final volume Of buffer equal to twice the approximate
settled gel volume and this suspension was slowly poured down the side of

a 1~1 x 100 cm Glenco analytical gel column to a fritted unit with a 500

m] reservoir. Packing was conducted at a hydrostatic head height of 100

cm for 4 hours. At this point excess gel was removed by aspiration to

give a final bed height of 95 cm and washing was allowed to continue for

24 hr under a 100 cm hydrostatic head.

Bi\°-Giel P-2 (-400 mesh) Chromatogaphy of Tryptic Digestion Products
Following tryptic digestion, samples were flash evaporated to dry-

“eSS and the residue was dissolved in a minimal volume of distilled

deionized water to give a final sample Of volume 0.3-0.5 ml.

This sample
Was loaded directly onto a 1.4 x 95 cm column of Bio-gel P-2 (—400

34

mesh) gel support and eluted with urea/Tris/mercaptoethanol buffer (pH
7.9, 115-120 uMhO) at a flow rate of 0.2 ml per minute directly into
liquid scintillation vials for a final sample volume of 0.4 ml/vial.
Sampl es were analyzed for [3H] and [14C] content following addition

of 8.0 ml of Formula C liquid scintillation mixture.

Construction of the High Pressure Liquid Chromatographic System

A high pressure liquid chromatographic system was designed following
an extensive modification of the procedure of Jones 31331. (63) to faci-
1itate analysis of the large number of samples encountered in this study.

A 1/16" bore 1/4“ 00 column Of length 40 cm fitted at one end with a

Steel frit and 1/16" 00 tubing was used as the column. A Milton Roy

(Milton Roy Co., St. Petersburg, Fla.) dual pump system and an Oil dia-
phragm pressure gauge (max psi, 2000) were placed on the inlet line of

the column system. The column tube was completely filled with dry

pel lionex SCX strong cation exchange resin (Reeve Angel Co., Clifton, NJ)
by slow pouring of the dry resin in fine granule form with constant
Vigorous tapping of the column tubing to insure uniform packing. Once
the column had been filled to capacity the inlet lines were purged of air
With the initial buffer system (0.05 M pyridinium acetate, pH 4.0), and
the inlet fittings attached to the column. Buffer was allowed to perco-

] a‘te through the support under pressure and extensive washing and equili-

bration Of the column was conducted at an inlet pressure of 300 psi at

amb 'i ent temperature.

p
wration of Buffers for High Pressure Liquid Chromatography

Pyridine was purified by distillation at one atmosphere pressure

35

u-

 

over ninhydrin. TO approximately 1.5 l of reagent grade, glass

distilled, pyridine in a 2 liter round bottom flask was added 5 g of
reagent grade ninhydrin. The flask was fitted to a glass water jacketed
condenser and heating mantle and the solution was heated until a mild
even boil was achieved. The first 10% (150 ml) of pyridine was discarded
and approximately 1200 ml was obtained as a constant boiling fraction at
the expected Tb of 113-116°C. The redistilled pyridine was stored at
room temperature in ground glass bottles wrapped in aluminum foil to
excl ude light.

The initial gradient eluent was prepared by adding 4.0 ml of pyri-
dine to 900 ml degassed deionized distilled water which had undergone
mil] ipore filtration over a 45 u membrane. The pH of the solution was
1:hen adjusted to 4.0 with glacial acetic acid and the volume brought to 1
1iter in a volumetric flask. The limit gradient eluent was prepared by
adding 160 ml Of pyridine to 800 ml water. The pH was then adjusted tO

6-0 and the volume brought to 1 liter.

H\iSLPressure Liquid Chromatographic Analysis of Tryptic Digestion
pr\Odusss

Following tryptic digestion, samples were flash evaporated to
d".Yness and then brought to a final volume of 120 ml with distilled
deionized water. Following adjustment of the pH to a value of 3.5 with
9] acial acetic acid, the preparation was pumped onto a 0.15 x 40 cm
C01 umn of Pellionex strong cation exchanger previously equilibrated with

0‘ 05 M pyridinium acetate, pH 4.0, at an inlet pressure of 200—300 psi.

36

Following a wash cycle of 5 minutes (two column volumes), elution was
effected with a convex gradient generated by a series arrangement of two
chambers containing 40 ml each Of 0.05 M pyridinium acetate, pH 4.0,
followed by a single chamber containing 40 ml Of 2.0 M pyridinium
acetate, pH 6.0 in a chamber rectangular Varigrad gradient mixer.
Fractions (2.0 ml) were collected directly into liquid scintillation
vial s and counted for [3H] and [14C] radioactivity content

fol lowing addition of 10 ml of Formula C scintillation fluid.

9&1 itative Analysis Of Tryptic Digest Products - Removal Of Urea from
the Bio-gel P-2 Resolved [3H1-Tryptg1han-Labeled Tryptic Peptides

Fractions corresponding to individual peaks of radioactivity eluting

 

from the Bio-Gel P-2 (-400 mesh) analytical column were pooled and the
Sal ts and urea present were removed by chromatography of the peptides on
a 0.9 x 20 cm column Of Bio-Gel P-2 (100-200 mesh) equilibrated with 0.2

M amonium bicarbonate pH 7.0. These fractions were later used to

\

establish the identity of the components isolated by the analytical

Bio-(;ei p—z (-400) urea column.

Individual components obtained above as well as from the high
pr‘essure liquid chromatographic analysis of the tryptic digests were

Co] lected and lyophilized to dryness.

I(ll\efltification of Resolved L-[3HLTryptophan-Labeled Tryptic Digestion

W

Identification of the resolved components Of the tryptic digestion

mixtures on each analytical system was accomplished by analysis via:

37

 

1. Tryptic digestion of L-[14CJ-tryptophan-labeled a 9_r_ B
globins (prepared by CM-cellulose chromatography) with [3HJ-L-trp-

labeled a 1151 B globins.

2. Paper chromatography of resolved [3HJ-tryptophan-labeled

peptides from each analytical system with standards of [14CJ-trypto-

phan-labeled a and 3 globin tryptic peptides by the method of Hunt,

._ --5

Hunter and Munroe (64).

“fl

Laper Chromatographic Analysis of Resolved Tryptophan-Labeled 8 Globin
Mic Digestion Products

Lyophilized samples from the high pressure liquid chromatographic
System or the P-2/urea system after removal of urea were dissolved in a

"'1' hi mal amount of water and spotted onto a 50 x 8 cm strip of Whatman 3

"m Chromatography paper. These chromatograms were equilibrated over the

o"‘Qanic phase of a mixture of n-Butanol, pyridine, glacial acetic acid

and water (90:120:18:72, volume ratios) for 24 hours. The chromatograms

Were developed in the decending direction with the aqueous phase Of the

abOwe solvent mixture. Chromatograms were dried for 24 hours at roan

temperature. The sample lanes were cut into approximately 100 strips,

”hi ch were of 0.5 cm length in the direction of chromatography and 1.0 cm

”1 de. These strips were placed in liquid scintillation vials, with 5 ml

of formula C liquid scintillation coctail and the radioactivity deter-

m‘i "ed.

wration of the Bio-gel P-2/Urea Chromatographic System for Double

I
%pe Analysis
Calibration of the Bio-gel P-2/urea chromatographic system for

d
ouble isotope analysis was accomplished by standard methods. The column

38

was run for 48 hours without a sample present and then 30 blank 0.4 ml

fractions were collected into plastic liquid scintillation vials. Nitro-
methane was used as the quenching agent and 5 sets of triplicate points
reFl ecting quench produced by 0, 2, 4, 6, 8 ul of nitromethane on 9,880
dpm of [MCI-toluene and 126,000 dpm of [3HJ-toluene were analyzed,
after addition Of 8 ml Formula C, by the method of automatic external
standardization as described in the Beckman LS-230 handbook (65b).
Quench was found to increase slightly as a function of time in plastic

vial s but sample homogeneity was maintained and the progressive increase

in quench followed the quench dependence with external standard value

throughout a 72 hr counting period.

Ca] ibration of the HPLC System for Double Isotope Analysis

Since sample volume and pyridine and acetate concentrations change

continuously due to generation of a gradient Of eluent concentration and

pH, a special calibration procedure was developed to account for the
e"b‘ll‘ect Of the changing sample composition during elution. A sham run of
the HPLC system was performed except no radioactive sample was included.
To alternate samples in plastic vials containing 1.0 ml formula C were
then added alternately either [3H]-toluene (1.26 x 105 dpm) or
E14CJ-toluene (9.88 x 103 dpm) and the S value was correlated with

the quench caused by the increasing concentration of the eluent compon-
eh"its. This relationship was found to be linear for [MC] and [3H]
q“ench and [14C] spill into the [3H] channel over the entire range

or S values encountered and was not identical to nitromethane calibration

(’1’ Samples containing either initial or limit gradient buffers. Quench

again increased slightly with time for plastic vials as compared with

39

Vi .-

glass but followed the initially determined quench dependence with
external standard S value for 72 hr. Decreasing S values diverged from
the dependence with quench established after this time period due to
extensive change in sample composition because of sample permeation of,
and evaporation from the plastic vials. This deterioration of the sample

necessitated determination of radioactivity within 48 hr of sample

pre paration.

Sena ration Of the Different Polysomal Size Classes by Sucrose DensitL
@dient Centrifuflion

Polysome profiles were prepared on linear 15-50% sucrose gradients
in Buffer A of Lodish 9131. (66), which contained 100 mM Hepes (pH 7.0
at 22°C), potassium chloride and magnesium acetate. Gradients were
POu red from a two chamber (20 ml each) gradient mixer with 17 ml of 50%
SUCrose in the proximal Chamber and 19 ml of 15% sucrose in the distal
chamber. Gradients were poured at 4°C and used within 12 hr of prepara-
t 1. 0n.

2X Buffer A was prepared (at twice the concentration specified by
LC><1ish gt al.) with distilled deionized diethylpyrocarbonate treated
water. TO 450 ml Of water was added 5.96 g Hepes (hydroxymethyl pipera-
Zi he 2-ethane sulfonic acid) 3.73 g potassium chloride and 0.429 9
magnesium acetate. The pH was adjusted to 7.0 at 22°C. The final
concentration Of Hepes, potassium chloride and magnesium acetate were
0 - 05 M. 0.10 M and 0.004 g, respectively. _

The initial gradient material was prepared by adding 15 g Of ultra-
put‘e sucrose to 50 ml 2X Buffer A, and this mixture was brought to a
vol ume Of 95 ml and agitated until the sucrose dissolved. Following

di Ssolution of the sucrose, 25 mg of cycloheximide, 5.0 mg sparsomycin

40

i.

and 205 pl of a solution of 9.74 mg/ml globin was added. The volume was
then brought to 100 ml and filtered through a disposable 22 u (Pore size)
mil lipore membrane prior to use. The limit gradient material was

prepared as above except 50 g of sucrose was used.

"‘1

1

41

seem;

Analysis Of the Products of the Reticulocyte Lysate System

Incubation of lysate derived from rabbit reticulocytes in the pre-
sence of hemin, GTP, ATP, creatine phosphate and creatine phosphokinase
and supplemented with potassium and magnesium resulted in rapid incorpor-
ation of [3HJ-labeled tryptOphan or leucine into TCA precipitable
material. To ascertain that this TCA precipitable material was in fact
authetic a and B globin polypeptides, analysis of the post ribosomal
supernatant was conducted. Globin polypeptides, labeled in the cell free
system as described in methods, were prepared by the acid-acetone method
of Ninterhalter and Huehns (56) and chromatographed in a CM-cellulose
column as described by Dintzis (55). The results of this procedure are
ill ustrated in Figure 1. It can be seen that over 97% of the total cpm
el uted from the column as expected for authentic a and 8 globins.
Recovery was typically 80-90% of the imput cpm. Very little material was
found to elute in the void volume or at high ionic strength at the end of

the elution program.

Incorporation of Radiolabeled Amino Acids into Peptidyl-tRNA

 

Incubation Of 1 ml Of the lysate and master mix components (above)
in the presence of 100 uCi of [3HCJ-tryptophan resulted in the incor-
poration of approximately 0.8-1.0 x 106 cpm into purified peptidyl--
tRNA- Figure 2 shows the material that was eluted from the Bio-gel P-10
desalting step. The peptidyl tRNA fraction was contaminated with labeled

sol uable globin to the extent of 20-40% of the total cpm eluting with the

42

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.

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H mc:m_c

43

 

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10203040506070

Fraction NO.

80

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45

 

 

 

 

 

 

 

void volume. Adsorption Of the void volume fractions onto a column of
DE-52 anion exchange cellulose followed by extensive washing removed all
detectable contaminating globin resulting in the elution Of a radiochemi-
cal ly pure fraction of peptidyl tRNA plus aminoacyl-tRNA corresponding to
a 60-80% yield on the basis of cpm obtained from the Bio-gel P-10 step
(Fig. 3). Subsequent concentration over a UM-2 ultrafiltration membrane
resulted in 5-10% loss of cpm to the membrane surface while no more than
2-3% was ever detected in the ultrafiltrate. This resulted in overall
yields with respect to total radioactivity from the P-10 chromatographic
step consistently greater than 55% but rarely higher than 75%. Radioac-
tivity which was associated with the ultrafiltration membrane was not
removed by inmersion in 6 M guanidinum chloride for 1 hour at 4°C and
hence seemed to be tightly bound to the membrane or associated with the
membrane in such a way that the solvent could not come into contact with
the bound radioactivity. It is unlikely that this loss of material
represents a nonrandom loss Of peptidyl tRNA since very heavily labeled
material obtained from a 5 mCi labeling of peptidyl tRNA and subsequently
concentrated on a small (0.8 ml) DE-52 column was subjected to size
distribution analysis by Bio-gel A 0.5m column chromatography directly
(without ultrafiltration) and this material gave essentially the same
Size distribution profile as an aliquot Of the same sample which was
further concentrated by ultrafiltration.
Fractionation Of tryptOphan-labeled nascent polypeptide material

d(‘ET‘TVed from peptidyl tRNA was accomplished on a Bio-gel A 0.5 111 column

e"l“ll'ibrated with 6 M guanidinum chloride 100 mM 2-mercaptoethanol as

47

 

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one as soon meet

.ce:_ou macenoxm cowcm Nmiuo m Ease <zmpipsecuqma co cow>eemn cowa=_m

m acumen

48

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am the SE
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351-13051)
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described in methods. Figure 4 shows a size distribution analysis of

L-[3HJ-trypt0phan-labeled nascent peptides representing a nascent pep-
tides labeled once, at the single tryptOphan residue a14 and a mixture of
a nascent peptides labeled once (Kd values greater than .55) or twice
(at Kd values less than 0.55) with L-[3HJ-tryptophan. The point of
insertion of the first a and 3 tryptOphan residues occurs at Kd 0.76-0.80
and the second 8 tryptOphan residue is encountered at Kd 0.55-0.60. The

reader may refer to Figure 5 for the locations of the key amino residues

referred to in this work. Analyses of this type have been conducted

previously by other workers (48,49) and characterization of such mixed on
and a nascent peptide size distributions was found to be essentially
independent of the choice of amino acid used to perform the labeling.
This is true to the extent that the different amino acids used to label

the nascent peptides have similar distributions and frequencies along the

Polypeptide sequences for the a and 8 globins. However, labeled amino

acids which are chosen to strongly weight one globin chain over the other
due to a relatively high frequency of occurrence of that residue in one
Polypeptide would be expected to enhance either the a or 5 components of

the mixed profile, and effect a qualitative change in the profile if the

‘1 Components differ from the 8 components.

gmbration of the Bio-gel A 0.5 m Column

Incubation Of either [14C]-Trp globins or [3HJ-Leu globins
with cyanogen bromide results in the two sets Of molecular weight markers

"hi Ch have been used in calibration of the analytical gel column for the

50

 

h

C. _ L — _ - ~ . I
0.00 0L0 0.No 0.UO 0-5.0 0.00 o.mo 0.3 Q 8 Ouinwo >0
D_m41mu~.mCi~1.oz oomnnoomziflu

.<zmo Pseeoeoa
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51

 

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relationship between molecular weight and elution volume. Since cyanogen
bromide cleaves the polypeptide backbone at methionine residues which are
found only once in each of the a and a globin chains (Fig. 5), incubation
of the globins with this reagent results in the production of 4 fragments
designated aCNBr1,2 and BCNBr1,2. An additional marker corresponding to
the unreacted a and a globins is also present. The use of [14CJ-trp-
labeled a and a globin results in two labeled cyanogen bromide fragments
of the a and B globin (aCNBrl, BCNBrl) facilitating their identification
and identification of the two C terminal fragments present in the
L-[3HJ-leucine-labeled globin digest which elute as unresolved compon-
ents. These products were used to check the calibration of new columns
and provide a comparison with calibration parameters obtained previously.
Figures 6 and 7 show the positions of the L-[14CJ-trp and

L-[3HJ-Leu labeled cyanogen bromide derived fragments from a and B

globin markers respectively as eluted during a calibration run of the
Bio-gel A 0.5 m analytical column. The markers are found to elute in the
expected order as demonstrated by Protzel and Morris (48) and the depen-
dence of molecular weight with the elution volume was found to obey the

0.555

ralationship of Kdo"333 vs. MN . Linear regression analy-

S‘is of the data shows that the points are well represented by the linear

equation:

IMO-333 = (-2.00869 x 10-3) Two-555 + 1.02576 r = -o.995
57 Ope and intercept parameters differ slightly from those determined by

p"‘01:zel and Morris (48) and Chaney and Morris (49) and represent the

pa I"ameters for this particular analytical column. This relationship

53

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wursogn :mmocaau xn muse—mm; mcwco_;ums u use 5 mg» mo mam>ompo an uwcemuno mmuvunmg ms»

.mmcmaamm mvpuamqapon cvnoFm a tea a on» c. mmsuvmmg v_ou ocean umuom_mm we =o_pouo_ mg»

m mgzmwu

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.mmE=~o> evo> new catapucv may xcms caguxmu mapn new mcvcmpmpxcmgqogu.:_o .cwaoFm
uzo Hgazuu new Hemzua mmuwuama muwsogn camocmxo .umpmnap-cmcqopqxgu-muefig .Pmchgmpuz
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56

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m? cowuspm mo gauge on» .mmu_uama muveoen cmmocoAu umpwnmp mcwuampumzmuub Lao; mg»

warm: mwmapmcm ucmsammnam a cw umcwsmxmmg mg: asspou a m.o < _mm-o_m as“ ea cowumgnppmu

N ogzmwu

58

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DISTRIBUTION COEFFICIENT

allows .
seating
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size.

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M‘

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allows determination of the mean molecular weight of the regions repre-
senting maxima and minima in the nascent peptide size distribution
profile and at the same time allows alignment of the position, on a given
nRNA molecule, of a ribosome carrying a nascent polypeptide chain of that

size.

Identification of Homozygous 8112 Val/Val Rabbits

 

 

In order to determine which components of the mixed nascent polypep-
tide size distribution were due to a nascent polypeptides and which are
chm to B nascent polypeptides, several experimental approaches were
adopted. The discovery by Shamsuddin gt_al. (65) of a rabbit B globin
polymorphism which results in the substitution of a valine residue for
the only a globin isoleucine residue providing an opportunity to directly
determine the a globin nascent polypeptide size distribution from a
L-[3HJ-isoleucine labeled radiochemically pure population of a peptidyl
tRNA. To obtain a reticulocyte lysate of this type requires the use of a
screening procedure to assess the ability of rabbit reticulocytes to
incorporate L-[3HJ-Ile into B globin chains as compared to incorpora-
tion into the three a ile position of the a chain. Three types of
results were expected. Homozygous 3112 Ile/Ile rabbits should
reflect levels of Ile incorporation into the a and B globins giving a
value of 3:1 for the observed a/B ratio. Heterozygotes in which half of
the 8 chains are of the phenotype 3112 Val would give a value of
331/2 or 6:1 for the extent of labeling of a relative to B globin.
Finally, the homozygous 3112 Val/Val phenotype should exhibit no

iSOleucine labeling of the 8 globin compared to normal levels of

60

‘se‘eucine
s:reening

each rabbi
:‘Tne label
[shaeffen
'iilO Of a
l. Diffic

m“
vy‘

..er in

isoleucine inCorporation (3 residues) into a globin. Since the usual
screening method involving CM-cellulose chromatography of the globin from
each rabbit, a procedure involving preliminary screening of the isoleu-
cine labeled globin samples by $05 gel electr0phoresis as described by
Schaeffer gal. (61) was adopted in order to expedite the analyses. The
ratio of a to B labeling with isoleucine for 8 rabbits is shown in Table
1. Difficulty in accurately counting the gel pieces accounts for the
scatter in the values for a/B obtained here as compared to the more pre-
cise values obtained from CM-cellulose chromatography which reflect the
expected values of 3:1, 3:0 and 6:1. These data indicated that samples 4
and 6 should be further analyzed by CM-cellulose chromatography for
L-[3HJ-ile incorporation into the a and a globin chains to establish
whether these samples are from 8112 Val/Val homozygotes. Figure 8

shows the results of (SM-cellulose chromatography of these samples. An
internal standard of [14CJ-Trp-label ed globins was included to insure
that the chromatographic system was working properly. It can be seen
that virtually all of the radioactive tritium present comigrates with the
‘1 910bin chains in both samples, confirming an assignment of both rabbits
to the phenotype of homozygous Val/Val 8112. A cell-free reticulocyte
protein synthesis system was prepared from pooled blood samples obtained

“‘0'“ both rabbits as described in materials and methods.

Went of a and Total Globin Synthetic Requirements for Potassium and

Ma "eSium

Simultaneous analysis of the rate and extent of incorporation of
L~ 1
E 4CJ-Ile and L-[3HJ-Leu into TCA precipitable material produced

by
a 8112 Val/Val lysate system as a function of Mg++ and K+

61

TABLE 1

Analysis of Globin Polypeptides for the Incorporation of
L-[3HJ-Isoleucine into a and B Globins by $05 Polyacrylamide
Gel Electrophoresis

 

 

Sample No. Cpm(a) Cpm(8) Rat10(a/B)
1 1506 494 3.0
2 1340 618 2.2
3 976 157 6.2
4 2750 228 12.1
5 1350 185 7.3
6 935 103 9.1
7 1257 378 3.3
8 1893 289 6.6
——-____
62

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.Auzmpg op gempv m .a m. cowuapm mo Lance ugh .m:_o:opomw pzozpwz mu_pamaa—oa :?Qo_m

u msu mcmucmc unauocoza _m>\_m> «Ham use .com_cmaeoo Loy mucmucmpm _mcgmucw mm umu:_ocp
mew mcwno_m m use a umymnm_ cmcnounacp muefiun4 .mummap muaoopaowumc Fm>\Pm> NHHm

mzomeoeo; a cw mcvoaopom_unzmu-4 saw: umpmnm_ newnopm yo xcqmemoumaogso mmopzp_mouzu

w mesmwu

63

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was carried out in order to ascertain whether the Optimal Mg++ and
K+ requirements for a synthesis and total (a and 8) synthesis were
similar or different. The result of such an assay is shown in Figure 9
for the dependence of the rate of incorporation of radioactivity into
(“[14CJ-isoleucine label) and total ([3HJ-leucine label) globin on
the Mg++ concentration in the presence of a final concentration of
140 mM KAc. Figure 10 shows the dependence of incorporation of label
into a and total globin on K+ concentration at the optimal Mg“+
concentration.

These studies indicate that the Optimal concentrations of K+ and
Mg++ for a and total globin synthesis are indentical and that by

inference the optima for 8 synthesis cannot be significantly different in

the reticulocyte lysate.

Labefling of 8112 (Val/Val) a Polysomes with L-L3H1-isoleucine

Specific labeling of a peptidyl-tRNA on a globin mRNA programmed
POIYSomes was accomplished by incubation of the 8112 val /val lysate
wittI L-{3HJ-isoleucine. Peptidyl-tRNA was prepared as described above
and IDeptidyl tRNA derived nascent polypeptide chains were fractionated
acc10r‘ding to molecular weight on a calibrated Bio-gel A 0.5 m column
equi] ibrated with 6 M guanidinium chloride and 100 mM 2-mercaptoethanol.
'ThE? Size distribution of radiochemically pure o-nascent polypeptides is
Shown in Figure 11. Table 2 lists the positions of the accumulation
maxima and minima for the a globin nascent polypeptides. It can be seen
that the size distribution is nonuniform and is comprised of at least 7

C .
onlbonents exclusive of the peak at Kd = 1 (derived from aminoacyl tRNA).

65

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mcwu=a_-mzmu-s .A----v 0=Po=a_0mw-musfiu-s .apam», assuopauwoae Fa>\_a> NHHQ
0;» c. umpmapm>m mm: cowamcucmucou ++mz co Am:_o=m~-m:mgu4v mmuoc u_pm;ucxm

=_no_m Am new av _muop use Amcpoampom_umuvfigaov :_no_m 5 05¢ mo mocovcmgmv use

a mesmwm

66

 

 

 

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Iampuhzmgaov genoPm quou use Am:_u:m_om_amu¢HH-4V cwno_m a any mo mucmucmawu one

OH ac=m_e

68

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70

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mo cem_cmqeoo powc_u mcwzop—m co_pm~:aoa mEomaPoa 05mm 05p :0 x_m:omcmppse_m empuaucou mm:
m:w_mnm_ o_aouom_ m_n:oo .uwnnmm _m>\pm> NHHm m mo mumm>_ m Eoce AIIIIV mmu_uamaaroq
:Pno—m Am van av _mpop umpmncp cacaopaxeuumzmgue use AIIIIV mmuwgamaapoa pcmomm:

cwno_m a um_mnm_ m:_u:m.omwumu¢nge yo mwmxflccm o_;qmcmopmeocco E m.o < _mmuowm NH mesm_e

72

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TABLE 2

The Positions of the a Globin Nascent Peptide Accumulations
as Determined by L-Isoleucine Labeling of 8112 Val/Val Lysates

 

0 Peak Kd MW Codon No.
I 0.22 15042.3 138-139
II 0.27 12754.3 116-117
III 0.35 9330.6 86-87
IV 0.43 6982.5 63-64
V 0.50 5242.1 47-48
VI 0.53 4510.5 41-42
VII 0.59 3557.4 32-33
VIII 0.66 2502.9 23-24
IX 0.73 1714.1 15-16

 

74

The most striking feature is the absence of a group of components in the
Kd range from 0.45-0.55 which is seen in nascent chain size distribution
profiles when other amino acids, such as tryptophan (see Figure 12), are
used for labeling, indicating that the large accumulation of nascent
chains in this Kd 0.45-0.55 region is due primarily to B nascent

polypeptide components.

Double Label Analysis of Nascent Polypeptides Using L-[14CJ—Isoleu-

 

cine and L-[gHJ-Tryptophan

 

To allow simultaneous comparison of the alpha globin nascent poly-
peptide size distribution and the mixed 0 and B globin size distribution
polysomes were labeled simultaneously with L-[3HJ-trypt0phan and
L-[14CJ-isoleucine. This choice of isotopically enriched amino acids
allows a direct comparison to be made between the a size distribution and
the composite total a and 8 size distribution. The tryptOphan labeled
mixed (0 and 8) size distribution will be represented as weighted by a
factor of two towards the 8 size distribution over the a size distribu-
tion component due to the presence of two tryptophan residues near the
N-terminus of the 8 chain in contrast with only one tryptophan residue
near the N-terminus of the a globin polypeptide. The result of such an
analysis is illustrated in Figure 12. The size distributions revealed by
the isoleucine and tryptophan labeled nascent polypeptides show striking
differences in the region corresponding to a Kd of 0.45 to 0.55 revealing
the presence of at least two large 8 components centered approximately at

Kd 0.47 and Kd 0.50. The remainder of the size distribution profile

75

shows a fairly similar though not identical distribution of components in

the a profile as compared to the composite a and 8 profile.

Characterization of the Bio—gel P—2 (—400 mesh) Analytical System for
Quantitation of L-[3H]-tryptophan-Labeled Tryptic Peptides

It was found that under the correct conditions of ionic strength,
and to a lesser extent pH, Bio—gel P—2 (-400) mesh gel filtration
separates the two 8 and one a tryptophan labeled tryptic peptides by a
mixed sieving adsorptive process. In this system, the three peptides
elute in order opposite to that expected for a separation based on
molecular size but in order of increasing basicity. Control
chromatographic analyses indicated that undigested globins elute with
the void volume. Figure 13 shows separation obtained following tryptic
digestion of a sample nascent polypeptides plus an internal standard of
L-[14CJ-Trp-labeled a and B globins. Separation of the 3T2 and 8T4
tryptic peptides was less reproducible than the separation between the
aT3 and the two 8 peptides. This system was used for analysis of the
extent of digestion of the sample and as an independent method for
confirmation of the ratio aT3 to 8T2 as determined by high pressure
liquid chromatography (below).

Identification of these tryptic peptides was accomplished by two
methods. Figures 14 and 15 show the result of chromatography of a
tryptic digest of [3H]-Trp-labeled a and 5 globin derived tryptic
peptides with either [14CJ-tryptophan-labeled a or 8 globins (pre-
viously isolated by CM-cellulose chromatography according to the method
of Dintzis). This procedure allowed immediate identification of the a

and two 8 tryptophan labeled tryptic peptides. Further identification

76

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we Leeee mew .6IIIIV muefiu .Auuuuv HIMQ .eceeeeeee esp meweee Feweeues eewueee

we mme_ meeweeem Lew wees we op cewuoeeeeo e mzewwe sews: eeeeceem Feceeecw ce eew>eee
ee meewpeme eeeome: eewmeew mzmg use new: eepmmmweaeo we: ewee_m .emwmeew xFEeewwce
.ceceeeexeeamuefiuue .weweepee mewueeexwee uceemec secw eecwepee mmeweeee owpexLu

cweewm ee_eee— emceepQALuamxmune we xseeemepeseeze caewee Acmee ooeuv Nae wemuewm ma meemwe

77

 

 

 

 

 

 

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FRACTION NUMBER

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caepee ewe—appeUIZU he eecweeee wee: mcweepm m ecu eueepmeepunzmH eeueeeeem

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ewueaeu cweepm e em_eeowucegeeuexupumzmuue we Agaeemeeeeeege Agmee ooeuv Nae _emuewm efi meemww

79

TI. 23 To...

 

 

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81

 

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was accomplished high pressure liquid chromatography with ultimate
reference to the established paper chromatographic system of Hunt, Hunter

and Monroe (64) (below).

Quantiation of the L-[EH]:tnyptophan-Labeled Tryptic Peptides by High

Pressure Liquid Chromatography.

 

The three tryptophan labeled globin tryptic peptides 0T3, 3T2 and
814 were resolved and quantified by high pressure liquid chromatog-
raphy on Pellionex SCX cation exchange support equilibrated initially in
50 mM pyridinium acetate pH 4.0. Figures 16 and 17 illustrate the‘
results of fractionation of a mixture produced by codigestion of
[3H]-Trp-labeled a or a globin nascent polypeptides with [14CJ-Trp
uniformly labeled a and 3 globins. The results of co-chromatography, in
separate experiments, of [14CJ-tryptophan labeled a and 3 globin
tryptic peptides with tryptic peptides derived from [3HJ-tryptophan
labeled a or'B nascent globin chains reveals that the first tryptic
peptide eluting is aT3 and that the latter two are the a tryptic
peptides.

Identification of the remaining 8 components as 8T2 and 3T4, was
accomplished by pooling of the appropriate eluate fractions of
[3HJ-tryptophan-labeled tryptic peptides from the Pellionex column
(Fig. 18). Following concentration by lyophilization, the samples were
subjected to paper chromatography as described in Methods. The results
of paper chromatography of a first and second tryptic peptides eluted
from a high pressure liquid chromatographic analysis is presented in

Figures 19 and 20, respectively. These results indicate that the order

83

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.cweewm e empeee_ ceseeuexguamxmuue ecu newee_m a use e ee—eee_uceceeuxguumoeflu-e
we meewpeee eweexgp mg» we mwmawece ewseeemeueeeege ewaeww eeemmece new: ma eeempu

84

 

 

 

 

 

 

 

 

 

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of elution of the tryptophan labeled tryptic peptides from the Pellionex
SCX system and the Bio-gel systems is aT3, 3T2 and 3T4.

Determination of the a and B Globin Nascent Peptide Size Distribution byg
HPLC of Tryptophan-Labeled Tcyptic Peptides

The HPLC system was used for the analysis of the a to 3 ratio in the
nascent chain size distributions. Figure 21a and b shows two
representative analyses of two different regions of tHE Bio-gel A 0.5 m
size distribution corresponding to Kd 0.26 and Kd 0.59. It can be seen
that the identification of 3T4 as the latest eluting tryptic peptide is
confirmed as this peptide becomes uniformly labeled later than the aT3
and 8T2 peptides, which are labeled together due to the nearly identical
positions of these tryptophan residues along the polypeptide at residues
a14 and 315, respectively. Figure 22 shows the abrupt rise in specific
activity of 3T4 tryptic peptide represented as the increase in the ratio
3T2/8T4 as a function of the distribution coefficient. Such analyses
were run on highly labeled ([3HJ-tryptophan) nascent peptides whose
size distribution is shown in Figure 23. Figure 24 shows the a and a
component size distributions as calculated from the value of aT3/BT2
obtained by HPLC analysis of tryptic digestion mixtures.

The validity of this method is apparent from the substantial agree-
ment between the a size distribution obtained from tryptic analysis with
the size distribution obtained directly by specific isoleucine labeling
of the u peptidyl tRNA fraction in the reticulocyte lysate (Figure 11).

Comparison of the a and 8 profiles indicates that, the a and the a

globin profiles are significantly different, as predicted from studies

94

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.mcweewm e ecu

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e~.o u ex .ueu

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95

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of isoleucine labeled a and mixed tryptophan labeled a and a nascent
peptides. The most striking difference is noted in the region from Kd
.55 to .45. At this Kd the a nascent polypeptides show a major accumula-
tion while very little of the a nascent components are present.

Also apparent is the relatively large a component in the region of
completed globin chains (Kd 0.18-0.26) relative to 3. This is consistent
with the observation of Protzel and Morris of an accumulation of u-globyl
tRNA relative to B globyl tRNA and extends their observation to smaller
nascent intermediates up to a Kd 0.26. This effect cannot be due to a
displacement in terms of the elution volume of a relative to a,
reflecting the fact that completed a globin is some 500 g/mol lighter
than a globin since such an effect would cause 8 components to be more
highly p0pulated at lower Kd's than 0 components, an effect which would
cause an underestimate of the a excess. Table 3 lists the distribution
coefficient for the major B nascent polypeptide accumulations obtained
from tryptic analysis of the tryptophan labeled size distribution for
comparison. Other regions of the a and a nascent polypeptide size
distributions reflect more similarity than differences between the a and

8 profiles.

Labeling of Reticulocyte Polysomes with N-Formyl Methionyl
Mat and L—[3H1-Methionine

The above analyses used selected amino acid residues as sites for
radiochemical labeling in order to provide as much information as possi-
ble about the nascent polypeptide size distribution for the a and a glo-

bin nascent polypeptides. The criterion by which a particular amino

104

TABLE 3

The Positions of the e Globin Nascent Peptide Accumulations as
Determined by Tryptic Analysis

 

8 Peak Kd MW Codon No.
I 0.28 12187.8 110-111
11 0.34 9579.9 87-88
111 0.46 6064.4 55-56
IV 0.53 4512.5 40-41
V 0.67 2343.6 21-22

 

105

acid was chosen was determined by the frequency and location of that
residue in the sequence of the protein being studied. Since each new
entry of a labeled amino acid residue into the growing nascent peptide
increases the specific activity of that polypeptide, distortion of the
size distribution profile can result. Since no information is obtained
concerning accumulations of nascent peptides prior to insertion of the
first labeled amino acid residue, it is also necessary to choose an
amino acid which is as close to the N-terminus of the growing polypeptide
chain as possible. To circumvent problems due to specific activity
changes and, more specifically, to obtain information about the size dis-
tribution of globin nascent peptides in the region of Kd 0.75-0.95, a
general method for the measurement of nascent peptide size distributions,
which is independent of amino acid sequence, was adopted. Radioactive
label was introduced into the N-terminal position of the nascent chains
by taking advantage of the observation that yeast initiator

tRNAfMat charged with methionine and formylated with a bacterial
aminoacylating/formylating enzymatic extract places a permanent N-formyl
methionyl residue at the N-terminus of the growing nascent polypeptides
in eukaryotic derived cell-free synthesizing systems (67). Fig. 25 shows
that the time course of incorporation of L-[35S] formyl methionine

into TCA precipitable material is linear throughout the first twenty
minutes of incubation. Figure 26 shows the result of an experiment in
which a and B globin nascent chains were labeled with L-[3HJ-tryptophan
and L-[35S] formyl methionine (f Met). These results reveal at least

one nascent peptide accumulation eluting at Kd 0.8 which is not apparent

in the tryptophan profile. Another component appears as a slight

106

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seew mcwceweumewwseewnmmmuie we coweueeeeeecw we muue mew me» we ecmEueemuez mm meemwe

107

 

30

TIME (min)

 

 

 

 

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eeemwe

109

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shoulder ahead of the free amino acid peak at Kd 0.92. Due to the pre-
sence of large amounts of free N-formyl methionine obscuring the small
molecular weight component appearing at Kd 0.92, independent verification
of this component was obtained by labeling polysomes with L-[3HJ-meth-
ionine. The L-[3HJ-methionine labeled size distribution is complex due
to the placement and subsequent removal of methionine from the N-terminus
as well as labeling of the internal methionine residues at Kd 0.48 and Kd
0.61. However, it has been shown that the N-terminal methionine is not
removed from the nascent globin chains until the nascent chains are at
least 20 amino acid residues in length corresponding to a Kd value of
0.65 (68). The results of such an experiment are shown in Figure 27. A
small molecular weight component is clearly demonstrated to elute ahead
of the free L-[3H] methionine peak, confirming the previous observation
obtained with [35S]-f Met. The composition of the two early nascent
chain accumulations in terms of the relative amounts of a and B globin
nascent peptides contributing to each was not determined. It is inter-
esting to note the apparent loss of the prominent B globin nascent pep-
tides in the region of Kd 0.45 to Kd 0.55 except for a very narrow
component at Kd 0.45. This is due to the removal of the N-terminal
methionine residues and the appearance of only the largest components of
the accumulation previously noted at Kd 0.45 to 0.55. The narrow compon-
ent at Kd 0.45 is due to labeling of a methionine residue 55 indicating
that the B accumulated B nascent chains in the region of the size profile
extending from Kd 0.45 to Kd 0.55 occur prior to and just past the 8

globin methionine codon 55.

111

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112

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Perturbation of the Nascent Polypeptide Size Distribution with
L-0-Methylthreonine

Several experiments are described which were designed to provide
information on the sensitivity of the steady state nascent peptide size
class accumulations to agents which might effect redistribution of ribo-
somal density along the mRNA molecule. L-O-methyl-threonine (L-OMT) is
an isoleucine isostere and is competitive with respect to isoleucine f0r
the isoleucyl tRNA charging reaction catalyzed by isoleucyl-tRNA synthe-
tase. Incubation of 3112 Val/Val reticulocytes or reticulocyte
lysate with L-OMT is known to induce a condition of isoleucyl tRNA star-
vation and inhibition of ribosomal translocation of isoleucyl codons (69)
of the a globin. Figure 28 shows the results of incubation of a
8112 Val/Val reticulocyte lysate with L-OMT, L-[14CJ-isoleucine
and L-[3H]-Tryptophan. The 3112 Val/Val phenotype ensures that
inhibition occurs only on a polysomes as has been demonstrated previous-
ly. Two peaks of shifted ribosomal density, which are observed by the
altered size distribution of the nascent a chains correspond to the
artifically induced reduction of ribosomal translocation rate at ile
codons 17 and 55. N0 peak is expected at isoleucine codon 10 since the
first isoleucine is placed at this point and hence no label appears in
the nascent peptides accumulated at this codon. A control size distribu-
tion pattern was determined using L-[3HJ-Trp and L-[14CJ- isoleu-
cine similar to that shown in Figure 12 in the absence of L-OMT. It is
important to note that the isoleucine label reflects the nascent peptide
accumulations caused on the 0 mRNA programmed polysomes whereas the
equally distorted L-[3HJ-Trp profile reflects mixing of the perturbed a
profile with the presumably unperturbed s profile. This contribution of

114

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meemwe

115

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the tryptophan labeled peptides is confirmed by the fact that subtraction
of isoleucine labeled size distribution profile from the total tryptophan

profile in both the control and L-OMT incubation results in residual

profiles which are essentially identical (Figure 29).

Perturbation 0f the Nascent Polypeptide Size Distribution with a Compli-
mentary Deoxyri bool qunucl eoti de

An assessment of the ability of a selected mRNA complimentary deoxy-
ri booligonucleotide to compete with and modify mRNA secondary structure
and thereby cause a perturbation of the nascent peptide size distribution
was conducted. The reticulocyte lysate of a 8112 Val/Val rabbit was
incubated with tetradeoxycytidylic acid at a concentration of 500 RM at
26°C and nascent polypeptides were labeled with L-[3HJ-trypt0phan. A
parallel control incubation was conducted in the absence of the tetranu-
cl eotide and labeled with LE14CJtryptophan. After termination of the
reaction with medium B plus cycloheximide and sparsomycin, the'[3H] and
[14C] labeled incubation mixtures were mixed and nascent peptides
Purified and analyzed as a single sample to evaluate the effect of the
tetranucleotide on the total a and B nascent chain size distribution.

A second experiment was performed in exactly the same manner except
that L-[3HJIle and L-[14C]Ile were used to label the lysates incu-
bated in the presence and absence of tetradeoxycytidylate (tetra C),
r‘espectively, to evaluate the effect, if any, of the oligonucleotide on
the a, nascent peptide size distribution. The rationale behind this

QXperiment is treated in detail in the discussion section.

119

Figure 30 shows the results of L-tryptophan labeling of the reticu-
locyte polysomes in the presence and absence of tetra C. This profile
reflects several shifts in the composite L-[3HJ-tryptophan labeled
nascent peptide size distribution as compared to the control. There is a
diminution of nascent peptides corresponding to Kd 0.28 and an increase
in components migrating on the gel column at Kd 0.40. A second shift
occurs in the region of lower molecular weight peptides. Nascent peptide
components migrating at Kd 0.70 and 0.73 are seen to be shifted to higher
molecular weight as reflected by a similarly shaped distribution of
compounds appearing at Kd 0.64 and 0.69. A parallel experiment involving
labeling of a polysomes with L-[3H] or L-[14C] isoleucine with and
without tetra C is shown in Figure 31.. As can be seen from this figure
there is no apparent difference in the control and tetra C treated
isoleucine labeled lysates indicating that no effect of incubation of
a-globin polysomes with tetra C is observable under conditions where the
L-[3HJ-trypt0phan labeled size distribution is effected. The B nascent
peptide size distribution was the only one significantly perturbed by the

presence of tetradeoxycytidylic acid.

Analysis of the Nascent Polypeptide Size Distribution as a Function of

 

Polysomal Size

Another approach used to define the effects of physiological levels
of mRNA ribosomal loading on the nascent peptide size accumulations
employed analysis of the nascent peptide size distributions present on
polysomes of various size classes. A 15-50% sucrose gradient containing

the elongation inhibitors cycloheximide and sparsomycin to prevent

120

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ribosomal run off during elongation and 0.1 mg/ml denatured globin poly-
peptides included as carrier material to insure against degradation of
nascent peptides was used. Figure 32 typifies the results of a number of
different experiments. Hhile size distribution of the smaller nascent
peptides looks essentially unaltered in the 3 size classes of polysomes
examined (dimers and trimers, tetramers and pentamers and greater) there
is a reproducible "filling in" of the nascent chain size distribution
profile in the region of the larger nascent peptide of Kd 0.4 to 0.5,
just to the 3' side of the large accumulation at Kd 0.35. A unexpected
result is the reproducible demonstration of a decreasing amount of
nascent chains of the size class corresponding to completed globin chains
as one examines polysomes of increasing size. This observation is con-
sistent with facilitated release or translocation of completed (or nearly
completed) globin chains on larger 0 polysomes, possibly due to a ribo-
some encountering reduced secondary structural interactions on the mRNA
of a polysome possessing a larger total ribosomal density. The degree of
reduction of completed chains on larger polysomes may be slightly under-
estimated since lighter polysomes (e.g., monomers, dimers) would normally
be expected to have a larger proportion of their numbers in a state of
relatively higher ribosomal density at the 5' end (smaller nascent pep-
tides) of the coding region. This effect is due to the increased proba-
bility that an inhibition which is translating at a decreased rate will
be located closer to the 5' terminus of the mRNA during the interval of
time necessary for a new initiation event to occur relative to uninhi-
bited ribosomes moving at a normal translocation rate. This effect is

most noticeable in dimers rather than in higher polyribosomes.

125

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126

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It is possible that the reduction of the largest nascent peptide size
classes up to and including completed globin is attributable to increased
ribosomal "run off“ as a function of increasing sucrose concentration, or
viscosity effects or distance traversed during sedimentation in the
gradient material. These seem unlikely since all size polysomes spend an
approximately equal amount of time out of the milieu of the lysate.

In order to further examine the effect of polysomal size and ribo-
somal density on the nascent peptide size distribution and to circumvent
the possibility of artifactual loss of nascent peptides during sucrose
gradient centrifugation, an alternative approach was used. The 8112
Val/Val reticulocyte lysate was incubated with 100 uCi of L-[3H]
isoleucine and a concentration of aurine tricarboxylic acid sufficient to
give 80% inhibition of protein synthesis (0.5 x 10’6 M) (Figure 33).

An incubation with 10.0 uCi of L-[14c1-isoieucine in the absence of

the inhibitor was conducted in parallel with the inhibited sample.
Following termination of the incubations with Medium B and cycloheximide
plus sparsomycin the samples were mixed and treated as a single prepara-
tion. The results of this experiment are presented in Figure 34. Incu-
bation of the lysate with aurine tricarboxylic acid is seen to cause a
relative increase in the amount of nascent peptides towards the high
molecular weight species including completed globin chains and especially
in regions of the prominent nascent peptide accumulations (at de 0.34,
0.50, and 0.57 relative to the accumulation observed at Kd 0.71. The
increase in the ratio of high molecular weight peptides relative to
smaller peptides on smaller polysomes is consistent with the results

obtained with inhibitors of elongation and the polysomal fractionation

128

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(below) but approach the phenomenon differently and extend the data to
very small polysomes, without exposure of the polysomes to any manipula-

tions not encountered during a standard peptidyl tRNA preparation.

Inhibition of Globin Translocation by Sparsomycin and Gougerotin

Inhibition of translocation to 28-30% of control by the elongation
inhibitors gougerotin, sparsomycin and cycloheximide was used to increase
ribosomal density on the mRNA in order to determine whether the degree of
ri bosomal loading of the mRNA would reflect a reduction in the nonuni-
formity of the nascent peptide size distribution due to an effect of
queuing of ribosomes along the mRNAs.

Preliminary studies of the dependence of elongation inhibition with
inhibitor dosage in the 8112 Val /Val lysate system was conducted
Ni th cycloheximide, sparsomycin and gougerotin. Levels of inhibitor
necessary to give approximately 70% inhibition of'protein synthesis were
determined for all three and the dose dependence established for
sparsomycin and cycloheximide (Figures 35 and 36). For the case of
Cycl oheximide use of L-[14J-isoleucine as well as L-[3HJ-isoleucine
eStablished that a globin synthesis was inhibited with the same dose
dependence as total (a plus B) globin synthesis (Figures 37-39). These
data were reexamined by incubation of the lysate with the amounts of
eye] oheximide, gougerotin or sparsomycin expected to give 70-75%
1'I'Ihibition as is depicted in Figure 40. All these data indicate that
this lysate responds, as expected, to the levels of the inhibitors
administered (70).

In subsequent experiments, isoleucine was used as the label so that

only the a globin nascent peptides were being considered with either

133

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L-[14CJ-isoleucine labeled nascent peptides as the control or
L-[3HJ-isoleucine for inhibited treatments. Sufficient gougerotin or
sparsomycin was used to give 70% inhibition of the overall rate of
protein synthesis. These reactions were terminated at 10 minutes by
addition of Medium B plus cycloheximide and sparsomycin. The reactions
were then mixed and treated as one sample throughout the steps of purifi-
cation and analysis of the peptidyl tRNA. Figure 41 and 42 show the
results of the sparsomycin and gougerotin incubations compared to a
cannon internal standard of [14CJ-label ed control nascent polypep-
tides. These figures illustrate that ribosomal density was increased
towards the smaller peptides but very little effect was observed on the
peak to valley ratios except for a reproducible filling in nascent
peptides at Kd 0.37 to 0.5 behind the prominent accumulation at Kd 0.34,
an observation which seemsito be a conmon characteristic of any treatment
which increases ribosome density on the nRNA or conversely slows riboso-
mal translocation (below). A change is noted in the region of completed
globins which may be due to slightly reduced effects of these inhibitors
on termination relative to elongation resulting in a depletion of com-
Pleted nascent peptides relative to slightly smaller sized nascent poly-'
Peptides. It should be noted however, that other studies have indicated
that, where examined, the peptidyl transferase activity and the peptide
termination activity of ribosomes exhibit similar properties including
the dose dependence of their inhibition with sparsomycin concentration
(711- This suggests that the decrease in high molecular weight nascent
Peptides on larger polysomes relative to smaller polysomes may be caused

507913 by increased polysomal size, a result that is consistent

146

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with the previous experiments.

An experiment was designed to investigate and compare the effects of

the inhibition of ribosomal translocation on different nascent chain size

distribution of different size class polysomes. Separate aliquots of

lysate were incubated in the presence or absence of sufficient sparsomy-
cin to slow translocation to 30% of control values. Incubations were
halted as usual, mixed and polysomes resolved, as before, by centrifuga-

tion on a 15-50% sucrose gradient. Parallel incubations of equal volume

were also sedimented separately providing uninhibited and inhibited poly-

some profiles as shown in Figure 43. As can be seen, the polysomal

profile of the sparsomycin inhibited reaction plus the uninhibited con-

trol profile when added together result in the observed mixed polysomal
Profile.

Polysomes of equal sedimentation value were obtained from the
Control and sparsomycin inhibited reactions and nascent polypeptides

Prepared from the pooled fractions as indicated. Figures 44, 45, and 46
Show the polysome profiles for low (dimers), medium (3-4 mers) and high

(5‘6 mers) polysomes. The control curves reflect the previously observed
dependence of decreasing of high molecular weight nascent polypeptides
wi th

increasing polysome size as expected. Added to this trend is an

1nc "eased filling in of the region from Kd 0.4-0.5 by the sparsomycin

""17 bited ribosomes relative to uninhibited ribosomes on polysomes of the

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p013'somes as observed above (Figure 41). An additional depletion in the

I" .
9910:: of completed globins is observed with Inhibited ribosomes

151

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compared to uninhibited polysomes of the same sedimentation value and
supports the possibility of a slight a differential effect of the

inhibitor on elongation relative to termination.

Thermal Perturbation of the Nascent Polypeptide Size Distribution
Since the studies with elongation inhibitors were designed to per-
turb the nascent peptide size distribution indirectly via the effect of
the ribosomes on the mRNA secondary structure, a complimentary approach
was sought which would allow perturbation of the nascent peptide size
distribution via a direct effect on mRNA secondary structure. This was
attempted by use of a temperature "shift-down" of steady state labeled
translating polysomes. First, to insure that reinitiation would occur at
lowered temperatures nascent peptides were labeled in an incubation
conducted at 15°C for 10 minutes (Fig. 47). Steady state labeling was
not attained; however, it appears that ribosomes were initiating and
slowly moving into the mRNA molecule. Peaks and valleys obtained corre-
Spond approximately but not exactly to those of the internal standard
incubated for the same time at 37°C. It is interesting to note that
"‘5 bosomal density is very high at the 5' region relative to the 3' areas
(Which have not become labeled) but the nascent peptide size nonuni-
1=°"‘m‘ity was not eliminated. Subsequent experiments were performed by
theme] "shift-down" of steady state labeled polysomes from 37°C down to
22°C or 15°C. For this analysis, separate aliquots (333 microliters) of
the 8112 Val/Val lysate containing exactly 100 uCi of L-[3H]iso-
1e“(tine or 30 uCi of L-[14CJ-isoleucine were labeled at 37°C for 5.5
mi nUtes and then the tritium containing sample was shifted down to the

aDDrOpriate temperature (15°C or 22°C) and incubated for another 4.5

161

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minutes before both reactions were terminated. The [3HJ-Ile labeled
samples (15 and 22°C) were then mixed with equal aliquots of the
L-[14CJ-isoleucine labeled lysate and nascent polypeptides prepared

as double label experiments. Analysis of the [3H/14C] ratio and

total radioactivity content in the purified peptidyl tRNA and nascent
polypeptides indicated that no differential loss of [3H] nascent Chains
occurred during the 37°C to 22°C shift and the 37°C to 15°C shift (Table
4). Results of the analysis of the size distribution fbr these samples
are presented in Figures 48 and 49. Thermal "shift-down” revealed a
progressive diminution of nascent peptides of size Class corresponding to
a Kd from 0.45 to 0.55, an increase of the relative amplitude of the
accumulation peak at Kd 0.35 and relative diminution of nascent polypep-
tides at Kd value Of less than 0.35. These observations are consistent
with increased hinderance to transloCation at regions of the a mRNA
corresponding to the Kd values of 0.60 and 0.35 with relatively little
effect elsewhere in the profile. These results are also consistent both
a decreased capacity of ribosomes to open hairpin structures and/or an
increased stability of certain hairpin structures at reduced tempera—
tures. As in the previous experiments, we cannot exclude other possibil-
ities which could result in a redistribution of ribosomal density along a
mRNA, such as depletion of one or more amino acyl-tRNAs or a relative
change in affinities of one or more tRNAs for the "A" site of the trans-
Iocating ribosome. It is known, however, that approximately 12% of the
total hyperchromicity of the globin mRNAs during thermal denaturation
occurs in the temperature range from 15-37°C (15) and the effect of a

Change in temperature on the stability of existing helical structures

164

 

 

 

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The Effect of K+ Concentration on the Nascent Polypeptide Size
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Optomization of lysates with potassium chloride or potassium acetate

yields quite different values for the translational rate optimum for
input of K+. KCl Optimization yields a maximal translation rate at

72 mM while KoAc raises this optima by nearly a factor of 2 to an input
K+ concentration of 128 mM. Preparation of nascent peptidyl tRNA was
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location or relative amplitudes of the nascent chain accumulation had
occurred as a result of increasing the input l<+ concentration by a

factor of 1.72 between the two salt optima for translation.

170

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Discussion

The rabbit reticulocyte contains predominantly two mRNAs coding for
the a and B polypeptide subunits of hemoglobin. Protein synthesized On
polysomes directed by these mRNAs accounts for approximately 95% of the
total protein synthesized in the cell, providing a and B globins in
nearly equivalent molar amounts.

Polysomes programmed for a and B globin synthesis contain the a and
a globin mRNA molecules and (on the average) 4 to 5 ribosomes pOpulating
a coding region of approximately 435 bases (429 for a globin, 442 for B
globin) which is known to be folded into extensive regions of secondary
structure in the uncomplexed mRNA molecule. Each translating ribosome
contains one tRNA molecule to which is attached one nascent polypeptide
intermediate in the synthesis of, in this case, an a or B globin polypep-
tide.

Investigations of Protzel and Morris (48) into the properties of the
population of nascent peptide intermediates involved in a and 8 globin
synthesis revealed that all possible nascent peptide sizes were not
equally represented in the pOpulation of nascent polypeptides. Analysis
of the size distribution of these intermediates revealed accumulations of
certain size classes relative to others in the population. Since every
ribosome contains one, and only one peptide Chain, and since the size or
molecular weight of the nascent polypeptide uniquely positions the
associated ribosome along the coding region of the nRNA, an accumulation
of a certain size Class of nascent peptide may be thought of as a reduc-
tion in the translocation rate of ribosomes in the region of the mRNA

corresponding to the length of the nascent peptide. The nonuniformity

173

 

of the size distribution of nascent peptides, then, indicates nonunifor-
mity in the local rates of translation through various portions of the
mRNA.

The rabbit reticulocyte lysate was chosen for the studies described
in this work in light of the extensive characterization of the a and B
composite nascent peptide size distribution already achieved in this
laboratory. The reticulocyte translational apparatus itself as well as
the products, a and B globin, and the a and B globin mRNAs comprise the
best Characterized eucaryotic cell-free protein synthesizing system
available. During the course of this work the primary sequences of the a
and a globin mRNAs have been determined by other laboratories (40,41) and
several hypothetical secondary structures for these mRNAs have become
available reflecting varying degrees of experimental support (38,72).

The initial problem involved resolution of the a and B globin
nascent peptide size distributions which contribute to the total globin
nascent peptide size distribution thereby allowing comparison of the size
distribution of two highly related, yet distinct, nascent polypeptide
populations which are being synthesized simultaneously by the same
biosynthetic apparatus. Analysis of such a system would not only serve
as a universal control for comparison of the two mRNA molecules and their
associated nascent peptides but would also serve as a starting point for
studies into the possibility of a and 8 mRNA structural interactions
reflecting some hither to unrecognized element(s) of coordinate control
of a and B globin synthesis. 1

Elucidation of the a globin nascent peptide size distribution in a
rabbit reticulocyte lysate system which is synthesizing both a and B

globins was accomplished by taking advantage of the observation that some

174

El.“

 

rabbits exhibit a polymorphism at position 112 of the a globin polypep-
tide which results in the substitution of a valine residue for the only
isoleucine residue present in the a globin polypeptide ( ). Reticulo-
cytes from rabbits homozygous for this trait incorporate no radioactivity
into B globin when incubated with radiolabel in the fOrm of L-isoleu-
cine.

Labeling of polysomes from such a rabbit results in a radiochemical-

ly pure population of nascent a globin polypeptides. Careful analysis of

 

this population in conjunction wfith internal molecular weight standards
reveals at least seven prominent accumulations of nascent a globin
peptides.

Comparison of the a chain size distribution with the tryptophan
labeled nascent Chain accumulations allowed tentative identification of
some regions of distinct differences between the a and 3 specific size
distribution. The large accumulation of peptide components from Kd 0.45
to 0.55 is seen to be present in the total labeling pattern but is not a
prominent feature of the a pattern. The large 8 specific accumulation
defines the low'molecular weight boundary of the most prominent feature
of the globin nascent Chain size distribution, that is, the large minima
observed at Kd 0.43. These data indicate landmark features of the 8
profile which contrast with the a profile. The size distribution of the
a chain profile is nonuniform but rises in a more continuous manner with
increasing molecular weight of the peptides.

‘ It is noted that some features of size distributions are functions
of the radiolabeled amino acid employed and the method of separation of
the nascent peptides. An incremental increase in the size distribution

profile is expected whenever a new radiolabeled residue is inserted into

175

 

 

the growing peptide chain, necessitating the Choice of an amino acid
which is located as close to the N-terminal of the growing polypeptide
chain as possible while maintaining representation in the protein
sequence at a minimum. Both isoleucine and tryptOphan fit these criteria
reasonably well. Secondly, the amplitude of the size distribution of
polypeptides of constant specific activity increases towards higher
molecular weight (or lower Kd value) due to the logarithmic properties of
the gel filtration process employed. That is to say, more members of the
possible 145 or so nascent polypeptides are eluted per unit volume at low
Kd than at high Kd. Finally, a peak in the size distribution profile is
to be expected at the Kd of the completed globins due to the absence of
components on the lower Kd side of the elution profile.

In order to determine the 8 size distribution in the presence of a
synthesis and at the same time circumvent the above mentioned artifactual
perturbations of the observed size distribution, two rapid, high resolu-
tion chromatographic systems were designed to permit accurate quantita-
tion of the tryptophan label at positions 14 and 15 of the a and B globin
chain respectively, isolated as the tryptic peptides aT3 and 5T2. This
experimental approach allowed quantitation of the relative contributions
of the a and B nascent peptides to the tryptophan labeled nascent peptide
size distribution profile.

These analyses produced an a profile which was essentially identical
to that observed wfith isoleucine labeling of 8112 Val/Val lysates.

The 8 pattern obtained revealed features which were quite different from
the a pattern. As expected, the large accumulations observed throughout -
the Kd range from 0.45 to 0.55 in the total size distribution are predom-

inantly a specific. The 8 pattern also exhibited reduced levels of B

176

 

 

specific nascent polypeptides compared to a throughout the region of the
elution profile corresponding to completed chains (Kd 0.20 to Kd 0.25).
This result was not unexpected as Protzel and Morris observed an excess
of a globyl tRNA (completed a globin still attached to tRNA) present on
polysomes, as compared to 8 globyl tRNA, measured by two entirely
different approaches. It should be noted that if the nonuniformity of
the nascent peptide size distribution does indeed reflect nonuniform
translocation rates then the size distribution profile provides a great
deal of detailed information about ribosomal flux through the entire
length of the mRNA coding region. However, as with most steady state
measurements, information is not available regarding the kinetics of
ribosomal movement through different regions of the mRNA, i.e., a
particular accumulation may reflect any number of changes in elongation
rate constants which result in either a net increase of flux into or 1
decrease of flux out of the region.

It is noted that any point of isotopic placement will result in an
increase in the amplitude of the profile to generate the low molecular
weight side of an apparent gaussian accumulation. This is seen in the
Trp and Ile size distribution profiles at a position corresponding to the
placement of the first residue (at Kd 0.80). This raises the question of
whether or not the accumulation at Kd 0.7-0.72 is real or simply an
artifact due to the initial incorporation of label into the unlabeled
growing nascent peptides. In order to investigate this possibility and
to implement a general method to accomplish labeling of eukaryotic .
nascent peptides at as few positions as possible and as close to the N
terminus of the nascent peptides as possible, L-[35S]formylmethionyl
tRNAfMet was synthesized and used to label the a and B globin

177

. '1

3““

 

nascent peptides. The f Met residues placed at the N terminus of the
globins are known to remain intact and are not removed during globin
synthesis (67).

Experiments utilizing methionine and formylmethionyl tRNA revealed
two nascent peptide accumulations which were undetectable by tryptophan
or isoleucine labeling indicating that they correspond to peptides of
length less than 10-14 amino acids. Labeling of polysomal nascent chain
N termini wfith [35$] fMet revealed at least one additional nascent
chain accumulation in the population at Kd 0.82 corresponding to a region
on the a or B globin mRNA centered at codon 9. Another component was
noted as a shoulder on the free amino acid peak at Kd 0.90. The latter
peak was confirmed by analysis of L-[3HJ-methionine labeled nascent
polypeptides which exibited considerably less contamination by free amino
acids in the Kd range of 0.85-0.95. The interpretation of additional
accumulation in the L-[3HJ-methionine profile between de 0.60 and 0.75
is complex and is probably due to removal of the N terminal methionine by
the reticulocyte processing apparatus since these aspects of the size
distribution profile are not reflected in the fMet terminally labeled
nascent chain population. However, it has been reported that the N-ter-
Ininal methionine residue is not removed from the growing nascent peptide
IJntil the nascent peptide has become approximately 25-30 residues long
(68). Furthermore, Rich gt_a_l_. (73) have observed that ribosomal struc-
tn1re completely protects the growing nascent peptides of less than 25-30
residues in length suggesting that the N terminal methionine is not
available for processing until the ribosome has moved past the twentieth
codon. Collectively, these data suggest that there are two additional
accunnilations in the nascent peptide size distribution of the a and/or 8

91°biri mRNAs in the vicinity of the ribosomal binding region. Kozak has

178

‘ T‘I

 

proposed that the 40S preinitiation complex which forms with reovirus
mRNA initially binds upstream from the AUG initiator codon and proceeds
to “scan“ the 5' noncoding mRNA sequence until it encounters the first
AUG codon at which point the 605 subunit binds to form the initiation
complex. Under conditions where secondary structure was weakened or
eliminated the 405 preinitation complex was able to bind to the mRNA with
comparable efficiency but was found to "scan“ into the mRNA coding
sequence, indicating a failure to bind the 605 subunit at the AUG codon
(74). This led Kozak to propose that elements of secondary structure are
necessary for 405 preinitiation complex recognition of the AUG initiation
codon and for prevention of 405 subunit "read through". Since, fbr
globin mRNA, the 405 complex is known to cover a much longer stretch of
nucleotides (80 nucleotides), as compared to the 805 complex (27-30
nucleotides) (75,76), an element of secondary structure which would
interact with the 403 subunit when it is located over the AUG initiation
codon would be expected to involve sequences near the 12-13th codon or
35-40 nucleotides to the 3 side of the AUG codon. Since the 80S ribosome
covers a smaller length of nucleotides, this putative secondary struc-
tural element would be expected to cause an accumulation of nascent
(:hains of a length anywhere from 7 to 10 amino acids long if the scanning
rnechanism and the mRNA structures supporting that mechanism are a general
feature of eukaryotic mRNAs, as has been suggested (77). Examination of
‘the N terminal formyl methionine nascent peptide size distribution
revealed two nascent peptide accumulations corresponding to peptides of
length 5-7 amino acids and 9-13 amino acids both of which retard the 805
ribosome and presumably would also retard a “scanning" 40$ preinitiation
co"IIPlex, preventing 40$ movement out of the ribosomal binding site and

thus laroviding the necessary situation for 608 binding to the 405 complex

179

 

 

while it is located.

The presence of peptide accumulations corresponding to slow local
translation rates past the first 9-10 mRNA codons is thus consistent with
evidence obtained by Kozak in support of a scanning model for 805 initia-
tion complex fonnation. The presence of secondary structure within the
405 ribosome binding site and its potential effect on the efficiency with
which 805 initiation complexes are formed emphasizes the possibility that
in cases where initiation is the rate determining step in protein synthe-
sis the actual limiting event in the initiation or preinitiation sequence
may provide a mechanism for differential control of mRNA expression at
the level of initiation.

Studies were conducted to evaluate the effect of ribosomal queuing
or the disruption of the normal distribution of ribosomes along the mRNA
on the nascent peptide size distribution. Incubation of the 3112
Val/Val lysate in the presence of L-0-methylthreonine caused L-0MT
redistribution of ribosomes on the a globin mRNA due to L-OMT induced
pauses at the isoleucine codons as a result starvation of the biosynthe-
tic apparatus for isoleucyl tRNA. This procedure grossly disrupts the
normal polysomal structure as can be seen from the Ile labeled elution
profile, but should have no effect on the B nascent chain profile. This
prediction was confirmed by formal subtraction of the L-[14CJ-isoleu-
cine from the L-[3HJ-tryptophan labeled profiles in a double label
experiment with L-DMT and comparison of the residual with that obtained
from subtraction of control (isoleucine and tryptOphan labeled) profiles
obtained in the absence of L-OMT. The results of difference analysis of
these profiles were found to be essentially identical, providing strong
evidence that the B nascent peptide profiles size distribution is

independent of the synthesis or the size distribution of the a globin

180

TI

nascent chain components.

Further studies centered on the effects of ribosomal loading of the
a globin mRNA caused by moderate inhibition of the rate of elongation
caused by the presence of sparsomycin and gougerotin. Under conditions
where a globin synthesis was inhibited to 30% of control synthesis poly-
somes containing a nascent peptides, as determined by Ile incorporation
in a 8112 Val/Val lysate, were shifted from a maximal size of 4-mers
and 5-mers to 7-mers and 8-mers. Comparison of the size distribution of

the a globin nascent peptides of inhibited lysates ([3HJ-Ile) to that

 

of an uninhibited lysates (preferred as an internal standard) showed an
increase in ribosomal density increasing towards smaller molecular weight
nascent polypeptides (the 5' end of the mRNA). However, this increase in
ribosome density produced by the antibiotics caused no significant Change
in the shape of the size distribution. It was originally thought that an
increase in ribosome density might reduce the effect of secondary struc-
ture by keeping the mRNA in an extended conformation. However, since no
decrease of the nonuniformity of the nascent chain size distribution was
created by decreasing ribosomal velocity, these data seem to indicate
that inhibited ribosomes are retarded to the same extent as uninhibited
ribosomes. Thus with the addition of elongation inhibitors, ribosome
density is increased on the mRNA molecule, while the amplitude of the
nascent chain accumulations increased in proportion to the ribosomal
density between the putative slow translation points (profile minima).
‘To investigate the effect of ribosomal density on the a nascent
peptide size distribution, the contribution of different size polysomes
to the total polysome profile was assessed. Polysomes labeled with
L-isoleucine and separated on a sucrose gradient were used to prepare a
globin nascent polypeptides for size distribution analysis. In these
analyses nascent peptides from very small polysomes (dimers) gave

181

 

essentially the same size distribution profiles as the larger polysomes,
in terms of the distribution coefficients of the characteristic accumula-
tions. There was, however, a very reproducible and continuous decrease
of nascent peptides of size corresponding to completed globin polypep-
tides with increasing polysome size. Since these polysomes were

separated on gradients which contained carrier denatured globin peptides

 

and large amounts of sparsomycin and cycloheximide it seems unlikely that t“

the higher molecular weight polysomes were preferentially losing complete

or near complete a polypeptides due to "run off" or degradation as

I

compared to smaller polysomes which had been in the gradient for the same
amount of time. It is possible that increasing sucrose concentration or
some other artifact encountered during fractionation of the polysomes may
have perturbed the system but it is not Clear why some nascent peptide
sizes would be effected differentially as compared to others. An inter-
pretation which is consistent with the decrease in high molecular weight
nascent peptides on polysomes of increasing size is that facilitated
release of ribosomes on the secondary structure of the region near the

terminator codon. The observation that a globyl tRNA was found in excess

 

on polysomes while B globyl tRNA was not may be due to the fact that a
globin is known to be synthesized on significantly smaller polysomes than
B polysomes. This suggests a possible cause and effect relationship
between the two phenomena. This result also suggests a possible rela-
tionship between the rate of initiation and the rate of release of
completed polypeptides at constant elongation rates, perhaps by interac-

tion of the 5' and 3' ends of the mRNA.

182

In order to study this phenomenon further, polysome size was reduced
by incubation of the reticulocyte lysate with the initiation inhibitor
aurine tricarboxylic acid. This experiment demonstrated that there was
a increase in the larger nascent a polypeptides relative to those of
smaller molecular weight on the ATA induced smaller polysomes. In addi-
tion, the relative amounts of accumulated a peptides increased on the ‘
smaller polysomes. Both these observations, as well as the apparent ,4
decrease of nearly completed a nascent peptides on larger polysomes, is F-
consistent with a ribosome induced unfolding of local areas of secondary 1
structure which thus facilitates translation of regions of the 0 mRNA
which have been freed from secondary structure, possibly by regions of
the mRNA far removed in sequence from one another, drawing support for
the possibility of long range effects of ribosomal loading on mRNA secon-
dary structure.

Studies were conducted to assess the sensitivity of the a nascent
polypeptide size distribution to changes in ionic strength. Reticulocyte
lysates have been shown to have different apparent K+ requirements
depending on whether acetate or chloride is present as the counterion.
This has been attributed to an inhibitory effect of chloride ion in the
system ( ). A lysate optimized with KCl and with KAc was found to
incorporate maximally at input concentrations of 72 and 132 mM, respec-
tively. Total tryptophan nascent peptide prepared from incubations at
either level of input K+ showed no significant differences in the
nascent peptide size distribution consistent with an absence of signifi-
cant effect of K+ concentration over this concentration range on mRNA
secondary structure or the distribution of ribosome density across the

mRNA. Changes in K+ concentration are known to produce changes in the

thermal stability of a number of mRNAs including the globin messenger

183

RNA as well as other natural and synthetic polynucleotides. The absence
of an effect here may be due to our inability to observe the dependence
of the nascent chain size profile with ionic strength values over a wide
enough range.

Amalyses of the a globin size distribution as a function of tempera—
ture were conducted under several different experimental conditions.
Incubation of the lysate at 15°C demonstrated that initiation proceeds at
this reduced temperature. Analysis of the nascent chain size distribu-
tion indicated that steady state labeling of the nascent chains is not
achieved at this temperature in 10 minutes. Nascent polypeptides towards
the 5' end of the mRNA were found to be heavily labeled but synthesis had
failed to proceed into the coding region of beyond that associated with a
Kd of 0.5 or less. Although ribosomes were densely populating the 5'
mRNA region, the size distribution was still nonuniform. This finding
could indicate that a tighter mRNA configuration is present at 15°C and
impedes ribosomes more efficiently due to lower rates of thermal "breath-
ing“ of secondary structure and also indicates the presence of more or
different structures at 15°C than are present at 37°C. It is known from
hyperchromism and circular dicroism measurements on globin mRNA conducted
as a function of temperature that 10-12% more secondary structure is
present at 15°C compared to 37°C. A shift in this distribution does not
seem to be due solely to slower ribosomal transit since inhibition of
elongation produced with gougerotin or sparsomycin had no such effect on
the nascent peptide elution prOfile.

Since steady state labeling had not been achieved under the above
conditions, further experiments relied on the transfer of polysomes which

had achieved steady state labeling of o globin with [3H] isoleucine at

184

 

 

37°C to an incubation temperature of 22°C or 15°C. Following termination
of the incubation as described in Methods the reaction mixture was
combined with [14CJ-Ile labeled nascent peptides synthesized at 37°C

as a control and internal standard. These experiments showed almost no
effect of the temperature shift to 22°C in the low molecular weight
regions of Kd values > 0.5. However, a redistribution of approximately
12% of the labeled nascent peptides occurred as an apparent loss of
labeled material from regions corresponding to Kd 0.35 to 0.4 with an
apparent increase in the prominent accumulation at Kd 0.27 suggesting
that ribosomes may be accumulating at a point of hinderance giving rise
to this accumulation as well as decreased flux past the point of accumu-
lation characterized at Kd 0.52. This interpretation is consistent with
an apparent "runoff“ of nascent peptide components corresponding to
molecular weights higher than the accumulation of Kd 0.27. The shift to
lower temperatures (15°C) from 37°C resulted in a pronounced effect on
ribosomal flux producing fine structure in the elution profile corre-
sponding to nearly gaussian accumulations at the Kd's corresponding to
accumulations seen at normal incubation temperatures but producing as
well as an increase of the effects noted at the first shift down tempera-
ture at Kd values less than 0.35. Again, these data contrast with those
obtained for lowered ribosomal transit rates at 37°C produced by gougero-
tin and sparsomycin in that a shift in the distribution coefficient of at
least 1 component was observed in the 37°C/15°C thermal shift experiment
demonstrating that ribosomal flux in specific regions of the mRNA are
affected by the lowered temperature which is not simply due to retarded
ribosomal translocation and is consistent with an effect of the known

increase in secondary structure for the globin mRNAs in vitro throughout

185

 

 

this (15°-37°) temperature range. He cannot, of course, exclude any of a
number of potential mRNA sequence specific effects which may be postu-
lated to have occurred. These effects include possible changes in the
association constant of an amino acyl tRNA for the ribosomal “A“ site or
amino acyl tRNA starvation due to a reduced aminoacylation activity or a
change in the growing peptide conformation any of which could modify a
local translocation rate constant. Thermal stabilization of helices is
the most plausible explanation for which considerable independent
physical data exists.

An alternative approach to the effect of mRNA secondary structure on
ribosomal translocation rate was designed to assess the effect of
sequence specific oligonucleotide binding in order to perturb the dynamic
structure of a translating mRNA molecule. 0f the limited possibilities
for a structurally complimentary oligonucleotide available to us, tetra-
deoxy C was chosen. This choice was dictated by the fact that the tetra
Cztetra G duplex would have sufficient stability to exist at a reasonable
concentration at 26°C and under the conditions of ionic strength approxi-
mated in the lysate by a minimal K+ concentration of 132 mM. The
sequence ~GGGG- occurs only twice in the coding region of the 8 mRNA and
does not occur at all in the a sequence, nor are there any G runs longer
than 4 in either mRNA molecule. This choice was also dictated by concern
over the known ability of complimentary oligonucleotides to bind to the
anticodon regions of certain tRNA molecules. The most probable instance
where this type of interaction could occur is the case of the proline
isoacceptor tRNA whose anticodon contains a tri G sequence. 0f the tri C
proline codons present in the a and B mRNAs, all 5 occur in the a globin

mRNA. Thus if the nascent peptide elution profile were to change due to

186

 

 

 

tetra C interference with some aspect of prolyl tRNA function, this
effect would be expected to be apparent in the a globin size distribution
at those places corresponding to tri C proline insertion points. The
presence of tetra C was found to have no significant effect on the a size
distribution compared to an internal control under conditions where
several significant effects were noted in the tryptophan labeled (a and
B) nascent chain size distribution. One of these effects is a shift in m-

the tryptophan nascent peptide accumulation at Kd 0.72 to an accumulation

.M'

of nascent peptides of larger molecular weight. This shift was totally 3*
absent in the a specific labeling pattern and furthermore occurred in a

region where neither mRNA codes fOr any proline residues. The observed

shift of nascent peptides occurred at a point corresponding to the

position of the first tetra G sequence in the 8 mRNA, the only oligo G

seqUence of greater than two residues in this region of either mRNA.

The simpliest interpretation of this result is that the high concen-
tration of the oligonucleotide served to perturb the equilibrium of a
hairpin structure which had given rise to the Kd 0.72 accumulations
shifting these accumulations to a new hairpin structure farther
downstream. The experiments discussed thus far were designed in an
attempt to gain insights into the origin of the nonuniformity of the
nascent peptide accumulations.

All studies using perturbative techniques are subject to many
criticisms due to the complexity of this system and hence do not
rigoroUsly prove a relationship exists between the translocation rate of
an RNA bound ribosomal complex and the secondary structure of the RNA
template. The results presented herein represent a contribution to a

growing body of observations consistent with the hypothesis that mRNA

187

secondary structure causes changes in the local rates of ribosomal
translocation. Clearly, independent confirmation must be sought for the
effect of RNA secondary structure on ribosomal translocation as well as
all other aspects of ribosome and ribosomal subunit-RNA interactions.

In this respect the work of Hurst gt_al. (32) and Pavlakis gt El:
(38) on the secondary structural analysis of the a and 5 globin mRNA
molecules using enzymatic probes of RNA structure provides one such
approach. The methodology developed by these workers relies on the use
of $1 nuclease as well as other nucleases to preferentially cleave
single stranded regions of mRNA molecules which have been labeled with
[32F] on the 5' end of the RNA molecule. This technique is performed
in parallel with sequencing reactions and thus allows the determination
of those nucleotide sequences which are single stranded regions. A
histogram representing the number and intensity of nuclease cleavage
sites of the RNA plotted as a function of Kd of the nascent peptide size
distribution analysis allows comparison of nuclease susceptible single
stranded regions with the nascent polypeptide size distributions. Figure
51 shows the 8 mRNA nuclease susceptible sites, as determined by Pavlakis
.gt_al. (79) correlated with the B nascent peptide size distribution.
Alignment of the nuclease accessible regions of the B nucleotide sequence
with the B nascent chain size distribution was accomplished by the use of
marker [14C] globin CnBr peptides used as internal standards and
[14CJ-ile labeled, L-OMT induced, nascent chain accumulations (see
Figures 8, 9 and 26).

It can be seen from Figure 51 that a significant number of the
nuclease cleavage points fall in regions of 8 profile minima providing

support for the hypothesis that ribosomes have an increased probability

188

 

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of being found immediately 5‘ to double-stranded regions in the mRNA and
that a relatively low probability exists (as reflected in a high translo-
cation rate) of ribosomes being found at the 5' end of single stranded
regions. The most significant correlation present in these data is that
between the highly nuclease susceptible region of the B globin mRNA as
shown by the numerous nuclease cleavages throughout 8 codons 50-56 and
the heavy accumulation of B globin nascent peptide components located
from Kd 0.45 to Kd 0.55. These data indicate that, in this case, accumu-
lated B nascent peptides correspond to ribosomal density immediately
adjacent to sequences which are inaccessible to single strand specific
nucleases and hence are at the 5' terminus of stable 8 globin mRNA struc-
ture, and at the 3' end of a stretch of single stranded structure. The
location of the B nascent chain accumulations is compared whth the
nuclease susceptibility data provided by Pavlakis gt al. in Figure 52.
Recently published secondary structure maps of the rabbit a and B 5'-ter-
minal regions, which extend into the coding regions of these mRNAs reveal
the additional correlation between nascent chain accumulations and mRNA
secondary structure alluded to in the discussions of tetra C induced per-
turbation of the B nascent peptide size distribution and fMet detected
"early" nascent peptide accumulation mapping in this region. The loca—
tion of these accumulations are presented in Figures 53a and 53b. It can
be seen in Figure 53b that tetra C interaction and destabilization of the
large B hairpin centered at codon 14-15 would be expected to shift the
accumulation to the next hairpin region at codon 24-25 as was observed.
Also mRNA structures predict the low molecular weight accumulations,
revealed by fMet and Met labeling of nascent chains, at points within and

slightly past the 403 ribosomal subunit and 803 ribosome protected

191

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194

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regions in both a and a globin mRNAs as discussed above.
The nascent peptide accumulations are consistent with secondary
structural elements ahead of the 7-8th amino acid position or 21-24th

codon nucleotide and the 10-16th amino acid corresponding to codon

 

nucleotides 30-45 and in essential agreement with the structures proposed
by Pavlakis gt al. for these regions. It must be emphasized that while

the nascent peptide accumulation at Kd 0.7 is known to be due to both a f’
and 8 components, the identity of the lower molecular weight accumula-

tions have not been determined and may be a and/or 8 in composition.

 

The studies presented here and previously support the view that mRNA
molecules have characteristic, nonrandom structures in terms of the loca-
tions and stabilities of helical or tertiary structural interactions. We
extend this hypothesis by proposing that portions of these structures are
present in the coding region of mRNA molecules effectively modulate the
rate of ribosomal movement through various regions of the mRNA. Data
presented here and by others support the hypothesis that the resultant
effect of secondary structure on stabilization of polysomal size and the
distribution of ribosomal density on the mRNA may effect a number of
aspects of mRNA function including initiation, termination and elongation
rates, and mRNA structural and functional half life. It should be noted
that several lines of evidence suggest that the nonuniformity of the
nascent peptide size distribution is not due to partial proteolysis of
nascent polypeptides. The size distribution profiles were unchanged when
labeling was COnducted in the presence of phenylmethylsulfonylfluoride
(80) or when excess denatured globins were added as carrier. Most

significant, however, is the appearance of at least two and possibly

196

three accumulations of nascent peptides of less than 30 amino acids (Kd
0.65) in the size distribution. Several workers have shown that peptides

of this size are completely protected from proteolysis by the 803

ribosomal structure as Cited above.

 

197

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5.

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7.

8.

9.
10.
11.

12.

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14.

15.

16.
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18.

19.
20.

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