Molecular Mechanisms of Protein Biosynthesis

719 Pages·1977·93.597 MB·English

by  Herbert Weissbach (Eds.)

MOLECULAR BIOLOGY
An International Series of Monographs and Textbooks
Editors: BERNARD HORECKER, NATHAN O. KAPLAN, JULIUS MARMUR, AND
HAROLD A. SCHERAGA
A complete list of titles in this series appears at the end of this volume.
Molecular Mechanisms
of Protein Biosynthesis
Edited by
HERBERT WEISSBACH
SIDNEY PESTKA
Roche Institute of Molecular Biology
Nutley, New Jersey
ACADEMIC PRESS New York San Francisco London 1977
A Subsidiary of Harcourt Brace Jovanovich, Publishers
COPYRIGHT © 1977, BY ACADEMIC PRESS, INC.
ALL RIGHTS RESERVED.
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Library of Congress Cataloging in Publication Data
Main entry under title:
Molecular mechanisms of protein biosynthesis.
(Molecular biology, an international series of
monographs and textbooks)
Includes bibliographies and index.
1. Protein biosynthesis. 2. Molecular biology.
I. Weissbach, Herbert. II. Pestka, Sidney. III. Se­
ries. [DNLM: 1. Genetics, Biochemical-Congresses.
W3I322 1976m / QH426 1103m 1976]
QP551.M62 574.l'9296 76-46464
ISBN 0-12-744250-2
PRINTED IN THE UNITED STATES OF AMERICA
79 80 81 82 9 8 7 6 5 4 3 2
List of Contributors
Numbers in parentheses indicate the pages on which the authors' contributions begin.
Nathan Brot (375), Roche Institute of Molecular Biology, Nutley, New Jersey
Charles R. Cantor (203), Department of Chemistry and Biological Sciences,
Columbia University, New York, New York
C. Thomas Caskey (443), Section of Medical Genetics, Departments of Medi
cine and Biochemistry, Kleberg Center for Human Genetics, Baylor
College of Medicine, Houston, Texas
Benoit de Crombrugghe (603), Laboratory of Molecular Biology, National
Cancer Institute, National Institutes of Health, Bethesda, Maryland
R. J. Harris (413), School of Pharmacy, South Australian Institute of Tech
nology, Adelaide, South Australia
C. G. Kurland (81), The Wallenberg Laboratory, University of Uppsala, Upp
sala, Sweden
Fritz Lipmann (1), The Rockefeller University, New York, New York
David L. Miller (323), Roche Institute of Molecular Biology, Nutley, New
Jersey
James Ofengand (7), Roche Institute of Molecular Biology, Nutley, New
Jersey
Maria Pellegrini (203), Department of Molecular Biology, University of Cali
fornia at Irvine, Irvine, California
Sidney Pestka (413,467), Roche Institute of Molecular Biology, Nutley, New
Jersey
Michel Revel (245), Department of Biochemistry, The Weizmann Institute of
Science, Rehovot, Israel
xi
XII List of Contributors
David A. Shafritz (555), Departments of Medicine and Cell Biology, and the
Liver Research Center, Albert Einstein College of Medicine, Bronx, New
York
Issar Smith (627), Department of Microbiology, The Public Health Research
Institute of the City of New York, Inc., New York, New York
G. Staffier (117), Max-Planck-Institut für Molekulare Genetik, Berlin-Dahlem,
Germany
Herbert Weissbach (323), Roche Institute of Molecular Biology, Nutley, New
Jersey
H. G. Wittmann (117), Max-Planck-Institut für Molekulare Genetik, Berlin-
Dahlem, Germany
Preface
In little more than a decade since the elucidation of the genetic code, it
has been possible to obtain considerable insight into the process of pro­
tein synthesis. There are, of course, many aspects of this problem still un­
explained, but yet enough information is now available to warrant publi­
cation of this volume. We hope that the reader will be made aware of
those areas that are still in a state of flux, and where relatively little infor­
mation is yet available. As the reader can see from the Table of Contents,
a wide scope of topics is covered. Transfer RNA, the initiation, elonga­
tion, and termination processes, ribosome structure and function,
mRNA translation, and DNA-directed in vitro protein synthesis are in­
cluded. A chapter on inhibitors of protein synthesis and one on the
genetics of the components are also included. An attempt has been made
to encompass both pro- and eukaryotic systems. Where possible, a uni­
form nomenclature for the various soluble factors is used throughout.
This should minimize the difficulties in reading the literature in this area
in which diverse designations permeate the published reports. Unfortu­
nately, in the area of eukaryote initiation, this is not yet possible.
It is hoped that this volume will be of value to a wide audience, not only
to those specializing in the field of protein synthesis, but to students and
nonspecialists as well. To achieve this goal introductions to the chapters
should provide an orientation and a guide to the material that follows.
The subjects, however, are covered in depth so that the reader should
have a basic knowledge of biochemistry and molecular biology. It should
be stressed that emphasis is placed on studies at the molecular level in
cell-free systems. Thus, in general, protein synthesis in intact cells, in­
cluding the related aspects of hormonal action, protein turnover, and
posttranslational modification, has not been included in this volume.
We would like to express our gratitude to the authors for the time and
effort expended in preparing their contributions to this volume. Their
cooperation in this endeavor was essential and was of great help to us. In
addition, we are grateful to Janet M. Hill, Bernadette McCaskey, Diane
Popowicz and Tanya Schulz for their technical help in editing and pro­
cessing the individual chapters. We would also like to thank the staff of
Academic Press.
Herbert Weissbach
Sidney Pestka
xiii
Twenty Years of Molecular Biology
FRITZ LIPMANN
For twenty years now we have been familiar with the double helix
structure of DNA. As is well known, its follow-up led to the chemical
explanation of genetic replication. The double helical structure has been
interpreted as being due to a pairing by hydrogen bonding between the
four side chain bases, guano sine (G) ^—* cy to sine (C), and adenine (A)
^—* thymidine (T) [or uridine (U) in RNA]. In the five to ten years after
its discovery, it became clear that the base-pairing mechanism repre­
sented the driving force in the whole sequence of reactions now known
under the name of genetic information transfer. To summarize briefly,
three main stages are distinguished: two transcriptions—(1) replication of
DNA in cell division and (2) transcription of DNA into an RNA—the
latter of which continues largely into the third phase, the translation of the
four-base code in the "messenger" RNA into the twenty amino acid ex­
pression in the polypeptide chain of proteins. In a rather complex manner,
this translation also turns out to involve a base-pairing mechanism of
A—»U and G—»C in triplet clusters between the messenger RNA and
amino acid transfer RNA.
This chemical definition of the mechanism of replication, which had re­
mained for so long a mysterious ability of living organisms, has changed
our way of thinking rather dramatically. However, so much attention has
since converged on the further filling in of the details of this information
transfer, that the novelty and excitement originally generated by the tran­
sition has now rather worn off. To some extent it was expressed in the
preference to call what we are dealing with molecular biology rather than
biochemistry. Therefore, I thought it might be worthwhile to preface this
book on protein synthesis by reviewing more soberly what actually is the
essential feature of this change, which one might consider, rather, a
change of attitude, and how it came about.
On an earlier occasion, at a meeting at Rutgers University on Organiza­
tional Biosynthesis, I entitled my introduction "Molecular Technology."
Although not quite clearly expressed in the talk, the main reason for
giving it this title was that I had come to the conclusion that molecular
biology is essentially a molecular technology. By this, I meant a chemical
1
2 Fritz Lipmann
technology where the working parts have the dimensions of angstroms
and microns rather than of the centimeters or meters as used in man-
designed technology. It was realized already then that to obtain a "minia­
turization" of chemical machines to the level of the size of the cell, one
has to use molecules as building blocks. And to do this, it appears one is
forced to work with "soft" systems rather than with the hardware system
of human technology. It would be shortsighted to attribute this change of
attitude exclusively to the discovery of the double helix. It was fortunate
that it went parallel with the general development of X-ray crystallog­
raphy, of which it was a part, and of electron microscopy. One could now
see the intracellular structures of which the biomolecular machines are
composed.
I think a consequence of the biomolecular revolution is that it led bio­
chemists to accept the essentially technological aspect of biochemical
mechanisms, which has had a most salutary influence on recent develop­
ments in the biochemical domain. This is in contrast to an attitude that I
remember from the early days of my studies in medicine, when we were
strongly warned against using "anthropomorphic" explanations for bio­
logical reactions, an attitude which we have now long thrown away and
have fared much better since doing so.
With this background, one might once more have a look at the actual
events during transcription and translation where the hydrogen bonding
through base pairing is the chemical means of information transmission. It
is rather straightforward in the transcription sector between polynucleo-
tides: the synthesis of a polynucleotide occurs on the template and forms
an antiparallel image of the template, in that the polymerase or replicase
finds first, by base pairing, a complementary preactivated nucleotide
triphosphate to be added next. This is then sealed to the 3'-hydroxyl
terminal of the growing polynucleotide chain by displacement of a pyro-
phosphate from a 5'-triphosphate annex of this particular nucleotide tri­
phosphate. The same sequence repeats until the chain is terminated.
From this example, one may generalize that in all cases the synthesis of
a polynucleotide image occurs by combination of (1) a placing mechanism
by hydrogen bonding of the newly to be added part of the chain which (2)
through use of energy available in the nucleotide triphosphate annex
when applied by pyrophosphoryl displacement for group transfer be­
comes the new terminal of the extending chain.
It was, indeed, a farsighted prediction of Crick to propose that an analo­
gous mechanism of base pairing also would have to apply in the transla­
tion step from polynucleotide sequence into the amino acid sequence of
polypeptides. As is well known, he did propose that for this purpose the
amino acid would be specifically sealed to an "adapter," a polynucleotide
Introduction: Twenty Years of Molecular Biology 3
SECOND LETTER
u C A G
uuu "^ ucu uHr~) υβυΊ U
Phe Tyr Cys
uuc J ucc UAC J UGC J C
UUA Λ UCA Ser UAA OCHRE UGA AMBER A
Leu
UUG J UCG UAG AMBER UGG Tryp G
7^~~
CUU ecu "^ CGU U
His
cue ccc CAC CGC C
Leu Pro Arg
CUA CCA CAA CGA A
IGIUN
CUG ^ CCG __ CAG CGG G
AUU Ί ACU AAU AGU ^| U
AspN Ser
AUC lieu ACC AAC AGC J C
Thr
AUA ACA AAA AGA Λ A
Lys Arg
AUG Met ACG J AAG AGG J G
~ ~
GUU GCU ] GAU GGU J U
| Asp
GUC GCC GAC GGC C
Vol Alo Gly
GUA GCA GAA GGA A
Glu
GUG GCG 1 GAG GGG J G
GENETIC CODE
Fig. 1 Code triplets for amino acids from Crick's introduction to the Cold Spring Harbor
Symposium, Vol. XXI, 1966; slightly revised, ochre and amber stand for termination
codons.
carrier of small chain length that contained amino acid-specific triplet se­
quences, and that the triplet sequence would then find its place on the poly-
nucleotide template. This template we now know to be the messenger
RNA (mRNA) translated from chromosomal DNA to guide the transla­
tion reaction, and the Crick proposition became reality when the mecha­
nism of amino acid activation by transfer to a tRNA was revealed. The de­
tails of this translation process are one of the topics of this volume and
thus omitted here.
I would like to expand, however, on one aspect of the code problem,
namely, the early prediction of the redundancy, or rather degeneracy as
physicists like to call it, of the code with regard to several nucleotide com­
binations (isotriplets) relating to the same amino acid. This is essentially
the result of the presence of twenty amino acids in all complete proteins.
The number twenty made a doublet code insufficient, as it would only
allow for 4 x 4, or 16 base combinations. Therefore, the need for a triplet
code was visualized early, and eventually when the triplet assignment was
completed, it turned out, as described in the Cold Spring Harbor Sympo­
sium of 1966, that all the 64 triplets available were occupied by placing of
amino acids or other functions. In other words, all of the possible 4 x 4 x
4 = 64 base combinations were being made use of. As shown in Fig. 1,
with only 20 amino acids available for this many combinations, nearly all
amino acids have several different code triplets to join with, all of the ones
4 Fritz Lipmann
Amino Number of Amino Number of
acid codons Codons acid codons
U
A
UU G Ala ' 4 GC I
Leu 6 U G
CU c U
AG Thr 4 AC I
G
A
AG
G U
Arg 6 U Val 4 GU I
CG c G
A
G U
U Gly 4 GG A
AG
c G
Ser 6 u U
UC Ac Pro 4 CC A
G G
Fig. 2 Amino acids with triplet redundancy of 6 and 4.
specifying for one amino acid being handled within a particular cell by one
specific aminoacyl-tRNA synthetase.
I found it interesting to order the amino acids in sequence of the abun­
dance of redundancy of their code triplets; this is shown in Figs. 2 and 3.
It appears that there are three amino acids with six, and five with four, dif­
ferent triplets. There is one amino acid that has three triplets; the largest
number are those that have two. At the other end of the scale there are
two amino acids that appear to have only one single triplet, namely, methi-
onine and tryptophan. It may be worth realizing that these two amino
acids are chemically the most complex compared with a general organic
simplicity in the side chain structure of other amino acids; they sometimes
Amino Number of Amino Number of
acid codons Codons acid codons Codons
lie 3 AU CU Cys 2 UGg
A
Asp 2 «Ü His 2 CAc1
Asn 2 AA« Lys 2 AA G
Glu 2 GA *
Met 1 AUG
Gin 2 CA *
Trp 1 UGG
Phe 2 UU«
{ UAG
Tyr 2 UA« iff JLfU UGAAA
Fig. 3 Amino acids with triplet redundancy of 3 to 1.