The initiation of protein synthesis

TIBS - July 1980

178 19 Hashimoto, S. and Green, M. (1979) Virology 94, 254-272 20 Fraser, N. and Ziff, E. (1978) J. Mol. Biol. 124, 27-51 21 Perricaudet, M., Akusj/irvi, G., Virtanen, A., and Pettersson, U. (1979) Nature (London) 281, 694-696 22 Chen-Kiang, S., Nevins, J.R. and Darnell, J. E., Jr. (1979)./. MoL Biol. 135,733-752 23 Green, M., Wold, W. S. M., Brackmann, K. H. and Cartas, M. A. (1979) Virology 97, 275-286 24 van der Eb, A. J., van Ormondt, H., Schrier, P. I., Lupker, J. H., Jochemsen, H., van den Elsen, P. J.

25 26 27 28

29

DeLeys, R.J., Maat, J., van Beveren, C.P., Dijkema, R. and deWaard, A. (1979) Cold Spring Harbor Symp. Quant. Biol. 44, 383-399 Wilson, M. C., Fraser, N. W. and Darnell, J. E. (1979) Virology 94, 175-184 Harter, M. L. andLewis, J. B.(1978)J. Virol. 26, 736-749 Akusj/irvi, G. and Pettersson, U. (1979) Cell 16, 841-850 Zain, S., Gingeras, T. R., Bullock, P., Wong, G. and Gelinas, R . E . (1979) J. Mol. Biol. 135, 413-433 Kozak, M. (1978) Cell 15, 1109-1123

The initiation of protein synthesis Tim Hunt

This article, the first o f a three-part series on protein synthesis, describes some o f the current ideas about the mechanism and control o f the initiation o f protein synthesis. The author's interests are in eukaryotic systems, but where there seem to be differences between the process in pro- and eukaryotes, or particularly striking points in prokaryotes these are mentioned.

30 Berget, S. M. and Sharp, P. A. (1979) J. Mol. Biol. 129, 547-565 31 Chow,L. T., Jewis, J. B. and Broker,T. R. (1979) Cold Spring Harbor Syrup. Quant. Biol. 44, 401-414 32 Dunn, A. R., Mathews, M. B., Chow, L. T., Sambrook, J. and Keller, W. (1978) Cell 15, 511-526 33 Klessig, D. F. and Anderson, C.W. (1975) J. Virol. 16, 1650-1668 34 Klessig, D. F. and Chow, L. T. (1980) J. Mol. Biol. (in press) 35 Klessig,D. F. and Grodzicker,T. (1979) Cell 17, 957-966

what these reactions are. It is quite possible to identify a protein synthesis factor, and to purify it to homogeneity, without knowing what it actually does in real life, and without having any very obvious way of finding out except by luck. We are just beginning to rectify this situation by raising monoclonal antibodies against each factor, and studying the effect of these reagents in crude cell-free systems, a process formally analogous to studies of mutants in those organisms where it is possible. I think we may be in for some surprises.

The problem

as a new set of equally specific associations Proteins are assembled on ribosomes in occurs, and so on hundreds of times for an ordered stepwise manner according to each protein made. This requires energy the sequence of bases in messenger RNA. supplied in the fortn of A T P and GTP; all Understanding this process requires told, it takes the equivalent of four high answers to many questions which fall into energy phosphate groups split to make one various categories. What are the natures of peptide bond, half of .this total being the steps in the overall process? What is the required to charge t R N A with amino acids, molecular mechanism of each step? What the other half being used for reactions catalyses these steps? Above all, what fea- occurring on the ribosome. The main question about the process of tures of the process give it its high accuracy, initiation is how ribosomes find the correct which is estimated as less than one mistake starting place on mRNA, and how the per 10,000 amino acids [1]? What controls process is controlled, for alterations in the the rate of protein synthesis? And so on. rate or nature of the proteins synthesized Even after more than 20 years of detailed seem mainly to occur at initiation. studies, many of these questions can only There are many possible approaches to be answered partially, and it is the purpose these questions, each with particular of this article and its two successors* to give advantages and disadvantages. In yeast and an up-to-date account of where we stand bacteria, genetic analysis may in principle now. be used to determine how many different The overall process of protein synthesis components are involved and, together can be divided into three stages (bearing in with biochemical analysis, what each mind the cyclical nature of the process): individual component does. In higher initiation, elongation and termination of the polypeptide chain. Throughout the eukaryotes, only biochemistry is possible at process a common recurring theme present, but it is an odd sort of biochemisemerges; at every step there exists a high try which relies heavily on the use of degree of specificity, implying a tight specific inhibitors of protein synthesis to association of two, or usually many more reveal the existence of intermediates, as components. Yet the very next step usually well as the more conventional purification requires that this tight binding be loosened and reconstruction approach. Particularly in the case of such a complex process as protein synthesis, simple biochemistry can (and has) misled the unwary, for, until the * R e v i e w s o n e l o n g a t i o n a n d t e r m i n a t i o n will a p p e a r nature of all the reactions is understood, in the n e x t t w o issues o f TIBS. Tim Hunt is at the Department of Biochemistry, Tennis appropriate assays cannot be devised; yet Court Road, Cambridge CB2 1QW, U.K. the purpose of the studies is to determine

Beginning at the end In both bacterial and eukaryotic protein synthesis, ribosomes can usually only bind to m R N A as separate subunits at different times, the small subunit joining the m R N A before the large one. When and how the subunits are separated after termination is not clear; most likely they separate as they get off the m R N A (it may even be essential for them to dissociate in order to disengage the message), and they are prevented from joining together by a protein which acts as an 'anti-association factor'; in bacteria it is called IF-3, in eukaryotes elF-3. This factor binds to the smaller ribosomal subunit and prevents the larger subunit from binding; under physiological conditions, the two subunits bind together with high affinity to form inactive 70 S or 80 S ribosomes if this factor is absent. Actually, eukaryotic cells usually contain a sizeable pool of vacant 80 S ribosomes which do not participate in protein synthesis unless they can be dissociated into subunits. This dissociation occurs very slowly compared with the rate at which the active ribosomes present cycle from subunits through polysomes and back to subunits again, though there are conditions under which almost all the ribosomes enter this inactive'pool - for instance, under various stressful circumstances - and equally rapidly leave it, as when cells are stimulated from a resting state to grow very rapidly. It is not clear what regulates these fluxes. The concentra© Elsevier/North-Holland Biomedical Press 1980

179

T I B S - July 1 9 8 0

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Fig. 1. The sequence of reactions in initiation. 40 S ribosomes produced by the termination step bind elF-3 to prevent them binding directly to 60 S subunits. Subsequently Met-tRNA ~ mRNA, and 60 S subunits join the complex in that order. Note that mRNA would normally have several ribosomes already engaged in its translation, so that where the diagram specifies 'mRNA ', this would normally be polysome structures; the resulting 40 S/polysome structures are readily detectable among the more conventional polysomes contaL~ing integral numbers of ribosomes. For simplicity' s sake, the factors that catalyse the attachment of mRNA are referred to as elF-4; in fact, at least three factors, 4A, 4B, and 4C have been resolved. Factors that play a catalytic role, rather than forming stoichiometric complexes are shown in parentheses.

tion of native subunits is, by contrast, rather constant, and is probably set by the concentration of eIF-3 in the cell, except when inhibitors have been added (both aurine tricarboxylate and poly I cause massive accumulation of subunits in reticulocyte cell-free systems). The question arises as to whether elF-3 participates in removing ribosome~ from m R N A after termination, or whether some termination factors bring about dissociation of ribosomes (and mRNA); most of the assays for termination measure the release of completed proteins rather than release of ribosomes from m R N A or their dissociation into subunits. The role of initiator tRNA In eukaryotes, the first step in the initiation pathway (Fig. 1) after the formation of native subunits is the binding of initiator tRNA [Met-tRNAO to the 40 S :mbunit, in a reaction that requires the initiation factor elF-2 and GTP (but which also proceeds in the presence of non-hydrolysable analogues of GTP). This is somewhat surprising, given that tRNA normally only binds to ribosomes at the di~rection of codons in mRNA, and indeed there was some resistance to the idea at first. The evidence for the non-codon-directed binding of Met-tRNAf is simple and compelling. First, 40 S ribosomes bind Met-tRNAf in the absence of mRNA, and second, they will not bind m R N A in the absence of bound Met-tRNAf. The situation in bacteria is not as clear cut, because the 30 S/fMet-tRNAf complexes are not so stable and m R N A seems to bind to 30 S subunits to some extent in the absence of fMet-tRNAf; nevertheless, in bacteria the presence of fMet-tRNAf greatly stabilizes the binding of mRNA, and some people have argued that bacterial ribosomes follow the same obligatory ordered binding as in eukaryotes. In eukaryotes, this step has

received particular attention as a possible site of regulation, since there are many circumstances in which initiation appears to be reversibly blocked at this step. Whereas it is usually possible to detect 40 S/MettRNAf complexes in cells or cell-free systems, they sometimes disappear, and when they do, protein synthesis stops. For example, this happens in reticulocyte lysates when they are either short of haemin, or when low concentrations of doublestranded RNA are added. The mechanism of this inhibition is the subject of intense debate; whereas it has been shown that these conditions lead to the phosphorylation of elF-2, and that protein kinases specific for this initiation factor are activated under these circumstances, there are inconsistencies in the story. There are also suspicions that other factors may play a part, such as oxidation of particular sulphydryl groups on the protein. It is also possible that the 'energy charge' of the cell may regulate the rate of initiation, for GDP has a 100-fold higher affinity for elF-2 than GTP, but does not allow the binding of Met-tRNAf. Binding the mRNA Once Met-tRNAf is bound (together with eIF-3, eIF-2 and GTP) the 40 S subunit is competent to bind mRNA. This requires additional factors, collectively termed eIF-4, and ATP; non-hydrolysable analogues of ATP, or ADP will not substitute. It is not known how much ATP is required, an interesting point, particularly since its function is unknown. Is one ATP hydrolysed per m R N A binding, or are many? Equally, the precise roles of elF-4a and elF-4b are obscure, though both are necessary for m R N A binding in fractionated systems. Early reports that they had ATPase activity have not been confirmed. So much for factors; the question of most

interest in this story is of how ribosomes find the initiation site on mRNA. The mechanism seems to be different in bacteria and mammals. In bacteria there is very good evidence that the 16 S ribosomal RNA plays a central role, by forming base pairs with a site on m R N A just to the left of the initiation codon, as first suggested by Shine and Dalgarno [2] purely on the basis of sequence analysis. Their conjecture is supported by several experimental findings. First, there is a good correlation between the extent of potential base pairing between particular mRNAs and ribosomal RNA and the frequency of initiation on that mRNA. More compellingly, an interaction between m R N A and ribosomal RNA was demonstrated directly in a classic experiment by Joan Steitz and Karen Jakes [3]. They formed initiation complexes between E. coli ribosomes and a 3W-labelled fragment of phage R17 RNA which contained the initiation site of the phage A protein. They next digested these complexes with colicin E3, an enzyme that specifically cuts 16 S RNA about 50 bases from its 3' end, where the putative interaction with m R N A was expected. Finally, they added SDS to dissociate the proteins from the RNA and ran the mixture on an acrylamide gel. The labelled m R N A ran as if it were hydrogen bonded to the rRNA fragment. Suitable controls ruled out various trivial explanations for the interaction. Analysis o f m R N A sequences and rRNA sequences.in eukaryotes has not revealed any :such corresponding complementarities, or any other regular features which might direct ribosomes to particular places on the mRNA. However, the ribosomes always seem to start at the first A U G sequence in the mRNA, in marked contrast to bacterial ribosomes which can select internal A U G codons to make a beginning. Indeed, bacterial ribosomes-can

TIBS -July 1980

180 initiate synthesis on circular (i.e. endless) messages, whereas eukaryotic ribosomes cannot, as Marilyn Kozak has elegantly shown [4]. The current orthodoxy, spelled out in detail in a review by Kozak [5], suggests that eukaryotic ribosomes bind at or near the 5' end of mRNA, using the 'cap', m 7 G 5 ' p p p X . . , as a guide. This accounts for the observation that improperly capped mRNAs are translated at a much lower frequency than their correctly capped counterparts, and that the translation of capped mRNAs is strongly inhibited by competitors such as m7GTP. However, there are messages lacking caps which serve as excellent templates for protein synthesis, such as E M C R N A which begins p U . . . and in which the initiation codon seems to be at least 400 nucleotides from the 5' end; how these messages work is unclear. Also unclear is which, if any, of the initiation factors recognizes the cap. A protein of Mr 24,000 has been isolated from ribosomes by affinity chromatography on m 7 G D P Sepharose [6], and this same protein can be crosslinked to m R N A when it is bound in initiation complexes, yet the protein does not belong to the canonical set of initiation factors, and was not recognized as being necessary for initiation when the factors were being isolated, purified and characterized, though it was probably present as a contaminant of eIF-3 and eIF-4B. It is suggestive that extracts of poliovirusinfected cells are bad at translating capped m R N A [7] (polio R N A is uncapped), and that the capacity of such a system to translate capped m R N A can be restored by adding a protein fraction containing this cap-binding protein (and not eIF-4B as first reported). It seems however that other factors may be involved, and it is not known what polio infection does to the 24,000 cap binding protein to inactivate it; indeed, it has not been shown directly that this protein is inactivated in these extracts. Work on this point is presently very active. The idea that eukaryotic ribosomes bind at the very start of the m R N A and work their way towards the first A U G codon, possibly using A T P as an energy source for such movement is supported by the observation that certain messages (tobacco mosaic virus R N A for example) which have a long stretch of nucleotides between the cap and the initiator A U G codon can bind more than one ribosome, one at the A U G and one to the left where no A U G exists [8]. This is made very obvious when edeine is added. This oligopeptide antibiotic is a highly specific inhibitor of initiation which allows 40 S subunits to bind m R N A while completely preventing the attach-

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Fig. 2. A simplified diagram of the E. coli RNA phages. (A ) shows a map of the three major genes, 'A "protein, coat protein and synthetase (the recently identified lysis protein is omitted for clarity; it overlaps the coat and synthetase genes in a different phase). ( B) shows the R N A - R N A interactions that seem to be significant for the control of translation, and ignores the very extensive base-pairing that occurs throughout the genome ~. It shows the postulated interaction between the start ofthe 'A' gene and residues about 870-880 downstream, and the interactions between the start of the synthetase gene and residues 1408 - 1432 lying upstream in the middle o f the coat gene. These interactions prevent ribosomes binding to the initiation sites of these genes, leaving only the coat protein open for translation in the early phase ofin fection. The synthetase gene is opened up by ribosomes engaged in the translation o f the coat gene, and when enough synthetase has been made, it binds to a site close to the start of the coat gene, indicated in (B), thereby stopping any more ribosomes binding to the RNA and clearing the RNA for transcription into the (-) strand shown in ( C). This (-) strand serves as a template for the synthesis of progeny (+) strands, which can be translated in their nascent state; ",4' protein can be made from these replicative complexes until the polymerase passes residue 875 or thereabouts, when the ribosome binding site is shut down by the base-pairing indicated in (B). Finally, as indicated in ( D), the ',4' gene is closed by this secondary structure, and although the synthetase gene is opened up by ribosomes making coat protein, the coat protein itselfbinds to the RNA at or near the initiation site of the synthetase gene, which is thereby effectively closed late in infection when sufficient coat protein has accumulated. This map is drawn to scale according to the data of Fiers et al., for MS2 TM.

ment of 60 S ribosomes to the 40 S/mRNA complex. In reticulocyte lysates, the presence of edeine leads to the formation of dimers and trimers of 40 S subunits bound to globin mRNA, and I believe that they stop at the initiation codon; but Kozak found larger numbers of 40 S subunits bound to m R N A in a rather more highly fractionated system and believes that the 40 S subunits can wander all the way down the message in the presence of this inhibitor. Whatever the case, in the absence of edeine but in the presence of inhibitors which arrest ribosomes at the initiation codon after they have synthesized one peptide bond (e.g. sparsomycin or diphtheria toxin), one finds additional ribosomes to the left of the one bound at the true start, but not to the fight, reinforcing the idea that they must enter at the 5' end of the message, and cannot bind inter-

nally without having 'walked' there. It is not known why or how bacterial ribosomes differ from mammalian or plant ribosomes to account for these differences in their behaviour.

Joining the 60 S subunit

The 60 S subunit probably joins the 40 S/mRNAIMet-tRNAf complex when the Met-tRNAf anticodon has engaged the initiator A U G codon, but this is not certain. The joining reaction requires an additional initiation factor called eIF-5, and entails a concerted set of reactions in which (1) the Met-tRNA~ becomes bound in the P-site of the 60 S subunit (i.e. it can form a peptide bond with the incoming amino acid or with puromycin), (2) the G T P which bound the eIF-2 and Met-tRNAf to the 40 S subunit is hydrolysed to G D P and Pi,

TIBS - July 1980

181

and (3) all the initiation factors leave the coat protein. A protein is apparently only extremely difficult if not impossible; all one ribosome [9]. Presumably the 60 S ribo- synthesised using nascent R N A molecules can say is that polysomes alter in size and some cannot join until after the elF-3 has in the replication complex as templates. number in ways that clearly indicate conleft, since this factor inhibits subunit These findings are accounted for by a trol at the level of initiation. association. Release of all initiat:ion factors model in which secondary structure in the absolutely requires GTP hydrolysis, and if R N A prevents ribosomes from binding to References non-hydrolysable GTP analogues are sub- either the A cistron or the polymerase cis1 Yarus, M. (1980) Prog. Nucleic Acid Res. Mol. stituted, the 60 S subunit cannot bind. tron, until a ribosome in the process of Biol. 23, 195-223 2 Shine, J. and Dalgarno, L. (1974) Proc. Natl. However, the details of this 'step', which making coat protein unravels the start of Acad. Sci. U.S.A. 71, 1342-1346 may in reality be composed of a series of the polymerase gene; but polymerase binds 3 Steitz, J. A. and Jakes, K. (1975) Proc. Natl. Acad. steps which we fail to distinguish owing to at or near the initiation site of the coat cisSci. U.S.A. 72, 4734--4738 lack of specific inhibitors (edeine is the tron, and coat protein at or near the start of 4 Kozak, M. (1979) Nature (London) 280, 82-85 only inhibitor of this step) are shrouded the polymerase gene (Fig. 2). In this way 5 Kozak, M. (1978) Cell 15, 1109-1123 6 Sonenberg, N., Rupprecht, K. M., Hecht, S. M. from us. Interestingly, bacteria lack an the RNA can be cleared of ribosomes so and Shatkin, A. (1979) Proc. Natl. Acad. Sci. equivalent of elF-5, and subunit joining that it can serve as a template for RNA synU.S.A. 76, 4345-4349 seems to proceed as soon as GTI' has been thesis, and the synthesis of A protein and 7 Rose, J. K., Trachsel, H., Leong, K. and Baltihydrolysed, very likely by the GTPase polymerase be kept well below the synmore, D. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, present in the 50 S subunit. thesis of coat protein which is required in 2732-2736 8 Filipowicz, W. and Haennis, A.-L. (1979) Proc. We are thus poised at the start of the far larger amounts than the others. Natl. Acad. Sci. U.S.A. 76, 3111-3115 message proper, awaiting the arrival of the Examples of such control in eukaryotes 9 Peterson, D. T., Merrick, W. C. and Safer, B. next aminoacyl tRNA and next month's are virtually non-existent. While it is clear (1979)J. Biol. Chem. 254, 2509--2516 account by Brian Clarke of what happens that different mRNAs differ in their 10 Lodish, H. F. (1975) i n R N A Phages (Zinder, N. next. intrinsic initiation frequency, as in the case D.ed.), pp. 301-318, Cold Spring Harbor of m R N A for ot and fl globin, which in- 11 Rosenthal, E. T., Hunt, T. and Ruderman, J. V. (1980) Cell 20 (in press) itiates at frequencies of 20 s and 12 s re12 Hers, W. et al. (1976) Nature (London) 260, Control of initiation spectively, there are few well-documented 500--507 Most of the control of protein synthesis cases of frequencies being regulated in rein both pro- and eukaryotes appe.ars to act sponse to specific signals. One or two exam- General reviews at the level of initiation. Such control is of ples seem to occur in early embryonic Revel, M. and Groner, Y. (1978)Annu. Rev. two general kinds which I shall call quality development, when a large excess of Biochem. 47, 1079-1126 control and quantity control. By quality largely inactive ribosomes is present along Grunberg-Manago, M. and Gros, F. (1977) Prog. NucleicAcid Res. Mol. Biol. 20, 209-276 control I mean the following: a situation in with a large population of mRNAs laid Jagus, R., Anderson,W. F. and Safer,B. (1980) Prog. which ribosomes, faced with a variety of down during oogenesis. There is beginning NucleicAcid Res. Mol. BioL (in press) different mRNAs, 'choose' to translate a to be quite good evidence that after activarestricted set of those messages in certain tion of the oocytes of frogs and clams, circumstances, whereas under other condi- which is accompanied by a general increase TIBS Reviews tions they translate a different set. Quan- in protein synthesis, changes in the pattern A n o t e to p r o s p e c t i v e a u t h o r s tity control refers to circumstances in which of protein synthesis without corresponding Most reviews in Trends in Biothe frequency of initiation on all messages changes in the m R N A population occur chemical Sciences are written at is raised or lowered across the board. Both [11]. The mechanism is not known, though the invitation of a member of the types of control have been found, and in it is widely believed that proteins associeditorial board. We do, nevertheboth cases one can point to specific ex- ated with mRNAs may specifically repress less, welcome unsolicited reviews. amples where plausible mechanisms have or enhance their translation. Modification If you are interested in writing for been proposed to account for them. The of such proteins either by allosteric or coTIBS, please send a brief outline, best examples of quality control are found valent mechanisms could be specified in accompanied by key references, to in bacteria, particularly in the regalation of such a way as to account for alterations in the Editor-in-Chief, or an approtranslation of viral mRNA. For instance, their activity. priate member of the Editorial The likely role of modifications of eIF-2 although the genes of R N A viruses like f2, Board. R17 and Qfl are present in equimolar con- activity in the general control of initiation If the proposal is acceptable and centration in infected E. coli, the., proteins has been mentioned earlier, but the field is does not overlap with a review specified by these genes are produced in currently in such a state of confusion as to already in progress we will send widely different amounts [10]. Studies of warrant a separate review of its own. As for you instructions for preparing the mutant viruses show that amber mutations possible alterations in other initiation facreview. near the start of the coat protein gene lead tors, very little is known apart from the case to a failure to synthesize anything except of poliovirus infection described above. My Corrigendum for the prematurely terminated N-terminal impression is that most people believe that Bailey, J. Martyn, TIBS, March, fragment of coat protein, whereas an eIF-2 is the main site of regulation, but that 1979, p. 68, the structure of amber mutation further along the gene the evidence for this belief is virtually arachidonic acid in Fig. 1 should leads to overproduction of polymerase and non-existent except in the case of reticulobe drawn with the double bonds normal synthesis of the A protein. In cell- cytes. Since most cells yield poorly active on the A5,8,11,14, positions free systems it has been found that coat cell-free systems (but nobody really knows rather than A4,7,10,13 as shown. protein shuts off synthesis of polymerase, why), it is difficult to analyse rate-limiting and that polymerase shuts off synthesis of steps, and analysis in i n t a c t cells is