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Annu. Rev. Cell Bioi. 1992. 8:197-225 Copyright © 1992 by Annual Reviews Inc. All rights reserved
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REGULATION OF TRANSLATION IN
Annu. Rev. Cell. Biol. 1992.8:197-225. Downloaded from www.annualreviews.org by ILLINOIS STATE UNIVERSITY on 11/17/12. For personal use only.
EUKARYOTIC SYSTEMS Marilyn Kozak Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854 KEY WORDS: protein synthesis, reinitiation, mRNA-binding proteins, mRNA structure, initia tion factors
CONTENTS 1 97
INTRODUCTION AN OVERVIEW OF TRANSLATIONAL REGULATION IN PROKARYOTIC AND EUKARYOTIC SYSTEMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
mRNA-SPECIFIC REGULATORY PROTEINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Repressor Proteins ..... .. . ......................... ...... ...... ... Activator Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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GLOBAL REGULATION OF TRANSLATION IN MAMMALIAN CELLS. . .. . . . . Phosphorylation of General Components of the Translational Machinery . . . . . . . . Targeting the Consequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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SELECTED ASPECTS OF TRANSLATION IN VIRUS-INFECTED CELLS .. . . . . . The Host ShutoffPhenomenon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Poliovirus Translation .............................................
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REINITIATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
FUTURE PROSPECTS .
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INTRODUCTION The striking differences between prokaryotes and eukaryotes in cell structure, in the mechanism of initiation of translation, and in the structure of messenger RNAs dictate strikingly different approaches to regulating translation. The first section of this review gives a thumbnail comparison of translational regulation in prokaryotic and eukaryotic systems. The remaining sections expand the discussion of regulatory mechanisms that operate during the initiation phase of protein synthesis in eukaryotes . 197 0743-4634/92/1115-0197$02.00
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In the field of eukaryotic gene expression there are at the moment three outstanding examples of translational regulation: (a) inhibition of ferritin translation by an iron-responsive mRNA-binding protein (IRE-BP); (b) modulation of translation via eukaryotic initiation factor-2 (eIF-2); and (c) induction of yeast GCN4 translation in response to amino acid starvation, a process that involves regulated reinitiation of translation and is a special case of regulation by eIF-2. These three paradigms, which have been elucidated in considerable molecular detail, are described herein at length , and some other emerging examples of translational control are considered briefly.
AN OVERVIEW OF TRANSLATIONAL REGULATION IN PROKARYOTIC AND EUKARYOTIC SYSTEMS A key difference between prokaryotes and eukaryotes concerns the basic mechanism whereby ribosomes engage the mRNA. Whereas prokaryotic ribosomes enter directly at the AUG initiator codon or at the nearby Shine-Dalgamo site (Calogero et al 1 988), eukaryotic ribosomes apparently enter at the capped 5' -end of the mRNA and advance to the AUG codon by linear scanning (Kozak 1989a). The prevalence of polycistronic mRNAs in prokaryotes vs monocistronic mRNAs in eukaryotes follows from that basic difference in the mechanism of initiation. Whereas the initial contact between bacterial ribosomes and mRNA requires no initiation factors or cofactors (Calogero et al 1988) , ATP and a considerable number of protein factors are needed for eukaryotic ribosomes to engage the mRNA (Hershey 1 99 1) . A further difference is that in prokaryotes the small ribosomal subunit can engage mRNA at the Shine-Dalgarno site before binding initiator Met-tRNA (Hartz et al 1 99 1 ) , whereas in eukaryotes, the small ribosomal subunit binds stably to mRNA only after initiator Met-tRNA has bound. Nevertheless, in both systems , actual recognition of the AUG initiator codon is accomplished primarily by base-pairing with the complementary anticodon in Met-tRNA , as evidenced b y the ability to force initiation from a non-AUG codon when the anticodon sequence is experimentally changed (Cigan et al 1 988a; Varshney & RajBhandary 1 990) . Other basic aspects of the initiation mechanisms in prokaryotic and eukaryotic organisms have been reviewed previously (Gold 1 988; Hershey 1 99 1 ; Kozak 1 983) . Between prokaryotes and eukaryotes there are more differences than similarities in the mechanisms whereby translation is regulated. In prokaryotic organisms (Gold 1988; McCarthy & Gualerzi 1990) , two mechanisms are widespread: (a) repression of translation by mRNA-binding proteins , of which at least 16 examples have been described (Andrake et a1 1 988; Dolan & Oliver 199 1 ; Kozak 1 988a) , and (b) regulation via switches in mRNA conformation that expose a previously sequestered ribosome-binding site. An excellent
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review by de Smit & van Duin (1990) illustrates some of the mRNA structures involved in this type of regulation. The conformational switches may be brought about by the binding of an activator protein (Altuvia et al 1991; Hattman et al 1991; Wu1czyn & Kahmann 1991), or by translation of the upstream portion of the mRNA (Asano et al 1 99 1 ; Berkhout & van Duin 1 985; Dick & Matzura 1 990; Mayford & Weisblum 1 989; Rogers et a1 1 99 1 ), or by endonuc1eolytic processing of the transcript (Dunn & Studier 1975), or by a switch in the start site for transcription (Ambulos et al 1 99 1 ; MacDonald et al 1 984; Schulz & Reznikoff 1 99 1 ) . In eukaryotes, on the other hand, there is only one documented example of a translational repressor protein (the IRE-BP mentioned above), no proven translational activator proteins, and no known regulation by switches in mRNA conformation. To the extent that activation of translation by a change in mRNA conformation might occur in eukaryotes, one would predict that it should involve exposure of the capped 5' -end of the mRNA-the apparent entry site for 40S ribosomes-rather than exposure of the AUG initiator codon. Regulation of translation by small naturally occurring antisense RNAs has been documented occasionally in prokaryotes (Citron & Schuster 1 990; Inouye 1988; Ma & Simons 1990; Wagner et al 1987), but not thus far in eukaryotes . [Anti-sense RNAs that have been detected rarely in eukaryotic cells apparently operate at a pretranslational level (Kimelman & Kirschner 1989; Skeiky & Iatrou 1990; Tosic et al 1990).] In prokaryotes, reinitiation may occur during the coupled translation of cotranscribed genes, but reinitia tion in prokaryotes apparently is not regulated; i.e. it does not become more or less efficient in response to changes in the intracellular environment. Thus regulated reinitiation, as discussed below for the yeast GCN4 gene, is unique to eukaryotic systems. While we can count one certain example of a translational repressor protein in eukaryotes and one proven instance of regulated reinitiation, both described below, the number of eukaryotic genes for which specific translational regulatory mechanisms have been defined is much, much smaller than the number of prokaryotic genes that can be so counted. I suspect the explanation is not that translational regulation is less common in eukaryotes, but that it often takes forms that are harder to recognize. For example, modifications of general components of the translational machinery, which are virtually unheard of in prokaryotes, are widespread in eUkaryotic cells; and, at least in the case of eIF-2, the modification impairs the ability of the factor to support protein synthesis. If the resulting global throttling of the translational capacity constitutes a selective regulatory device, however, it would have to be targeted somehow--if not to specific mRNAs, then to groups of mRNAs. Some speculations about how that might occur are offered below. The possibility that translational control, especially in developing systems , might involve
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compartmentalization of mRNAs and/or components of the translational machinery is also discussed, although the available data are inadequate to resolve the issue. The possibility of regulation at the level of polypeptide chain elongation has been considered elsewhere (Ryazanov et al 1 99 1 ) . The mechanisms mentioned above effect switches i n translation, which i s the way translational regulation i s usually defined. But the definition might be broadened to include a substantial number of mRNAs that have, built into their invariant structures, features that force the standard translational ma chinery to perform in nonstandard ways-- initiating translation from two sites, for example, or shifting to a different reading frame during the course of elongation, or translating through a stop codon. Those aspects of translational control are discussed in other reviews (Atkins et al 1 990; Kozak 1 99 1 a).
mRNA-SPECIFIC REGULPJORY PROTEINS Repressor Proteins In mammalian cells, synthesis of the iron-sequestering protein ferritin is regulated by iron availability (Klausner & Harford 1 989). The regulation is accomplished by a �90-kd protein that inhibits translation by binding near the 5'-end of ferritin mRNA (Figure la). The target site for the repressor, a conserved stem-and-loop structure depicted in Figure Ie and d, is called appropriately the iron-response element (IRE), and the repressor protein is the IRE-binding protein (IRE-BP). A recently obtained full-length cDNA clone for murine IRE-BP reveals that the repressor is related to mitochondrial aconitase , and enzymatic tests reveal that IRE-BP itself has aconitase activity (Kaptain et al 1 99 1 ) . The fact that aconitase contains a labile [Fe-S] complex suggests a likely mechanism whereby IRE-BP senses iron levels. As illustrated in Figure 1 , the reversible binding of IRE-BP to mRNA is thought to reflect reversible oxidation of a critical sulfhydryl group: when iron is scarce, the reduced [3Fe-4S] form of the protein binds the IRE; when iron is abundant, the oxidized [4Fe-4S] protein dissociates from the IRE (Haile et al 1 989; Rouault et al 1991; Constable et al 1992). The identification of IRE-BP as a cytosolic aconitase offers interesting insight into how a eukaryotic repressor protein evolves. One is reminded of the situation in prokaryotes, where virtually every translational repressor protein has a primary function other than regulating translation (Kozak 1 988a)! The effects of IRE-BP on ferritin translation were deduced from model reactions between IRE-BP and mRNA fragments (Rouault et al 1 98 8; Barton et al 1 990; Constable et al 1 992), from the effects of IRE mutations on translation in vivo (Caughman et al 1 988), and from direct studies of translation in vitro (Brown et al 1 989). Qualitatively those results support the model in Figure la, but the range of induction in some test systems is
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(a) Regulated Translation ot Ferritin mRNA
(b) Regulated Stability 01 Transterrin Receptor mRNA
(c) Structu,e of Fe,ritin IRE GU
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Annu. Rev. Cell. Biol. 1992.8:197-225. Downloaded from www.annualreviews.org by ILLINOIS STATE UNIVERSITY on 11/17/12. For personal use only.
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Post-transcriptional regulation of two genes involved in the uptake and detoxification of iron in mammalian cells. Regulation is accomplished by the interaction of a protein, IRE-BP (shown as a triangle), with a specific target sequence IRE (shown as a hairpin in the mRNA). The total amount of IRE-BP in cells is invariant, but the affinity of the protein for its target site varies with the iron status ofthe cell, as explained in the text. (a) Translation offerritin, a protein that sequesters excess iron, is inhibited by the binding of IRE-BP to ferritin mRNA. (b) IRE-BP binds to and stabilizes the mRNA that encodes transferrin receptor ( TfR) , a protein that promotes the uptake of iron into cells.The 3'-noncoding sequence of TfR mRNA contains five IRE motifs, only one of which is shown. The presumptive target sites for ribonuclease(s) near the 3' -end of TtR mRNA have not yet been mapped precisely. The actual sequence of one IRE (c) is shown along with a consensus IRE structure (d) derived from phylogenetic and other data (Hentze et al 1988; Leibold et al 1 990).
considerably less than what has been observed with endogenous ferritin genes, which suggests that control of ferritin synthesis might occur at multiple levels (Mattia et al 1 989; Goessling et al 1 992). While translation can be inhibited by base-paired structures per se (Kozak 1 986a, 1 989b), the particular stem-loop structure that constitutes the IRE is not very stable, which probably explains why it does not inhibit translation on its own. The binding of IRE-BP may stabilize the base-paired component of the IRE (Harrell et al 1 99 1 ) . In ferritin mRNAs the IRE is located typically about 30 nt from the cap \ and this 5' proximal location is very important. In a telling experiment, repression was lost when the IRE was repositioned 67 nt from the 5' -end of a test transcript (Goossen & Hentze 1 992). The simplest interpretation is that a 40S ribosomal subunit, once bound to the 5' -end of the mRNA, can displace proteins as it advances, much as the scanning 40S ribosome has been shown to melt certain base-paired structures (Kozak 1 986a, 1 989b). Given the apparent ability of the 40S ribosomal subunit to clear a path as it advances, IThe IRE is often part of an extended stem that actually begins mRNA (Wang
et al
1990).
-10 nt from the 5'-end of the
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an mRNA-binding protein (or a hairpin structure) will inhibit translation only if it is close enough to the 5 ' -end to prevent the initial binding of the 40S ribosome. It is intriguing that the same protein that down-regulates ferritin synthesis when iron is scarce has been found to up-regulate production of the transferrin receptor (TfR) protein, as illustrated in Figure lb and described more completely by Klausner & Harford ( 1 989). The opposite effects of iron deprivation on ferritin and TtR synthesis reflect the location of the IRE in each mRNA: masking by IRE-BP of the 5' -end of the mRNA understandably inhibits translation of ferritin, while masking of the 3 ' -noncoding end of the mRNA , which is irrelevant to ribosomes but is often the target of ribonucleases (Peltz et al 199 1 ) , increases the stability of TtR mRNA. Recent data suggest that IRE-BP also regulates the translation of erythroid 5' -aminolevulinate synthase, the first enzyme of the heme biosynthetic pathway (Cox et a1 1991; Dandekar et al 1 99 1 ). Thus a single remarkable protein controls both iron homeostasis and a key metabolic pathway for iron utilization. The search for other eUkaryotic genes controlled by translational repressor proteins has produced no compelling results as yet. A few candidates merit watching (Kaspar et al 1992; Lee et al 1991; Schafer et al 1990), but the evidence adduced so far is barely suggestive. The mere demonstration that one or another protein binds to an mRNA leader sequence permits no inferences about effects of the protein on translation. "Masking proteins" are a traditional explanation for some aspects of translational regulation in developing systems (Richter 1 99 1 ) , but the cis-acting elements underlying such control are often at the wrong end (i.e. the 3' rather than the 5'- end) of the mRNA, and no one has yet shown that the mRNA-binding proteins have the predicted effects on translation. The key experiments would be to show that (a) with native ribonucleoproteins (mRNPs) as templates, the pattern of translation in vitro reproduces the restricted pattern observed in vivo; (b) extraction of proteins from mRNPs specifically activates translation of the mRNAs that were restricted in the preceding step; and (c) re-addition of a purified protein fraction inhibits translation of the specific target mRNAs. It is the last step that has failed in every attempt to test the masked mRNA hypothesis (Grainger & Winkler 1987; Standart et aI 1 990). Thus while there is overwhelming evidence that certain mRNAs are present in oocytes prior to the onset of their translation, the evidence implicating soluble translational repressor proteins is not overwhelming. The fact that mRNAs differ in the extent to which they accumulate in mRNPs has been cited as evidence that RNP proteins recognize specific mRNAs (Kelso-Winemiller & Winkler 1991 ), but an alternative interpretation is that mRNAs that cannot engage ribosomes (e.g. due to secondary structure near the 5' -end) are left exposed and therefore pick up RNP proteins. In other
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words, accumulation in mRNPs might be the passive fate of untranslated mRNAs rather than a mechanism that actively restricts translation. In view of the ubiquity of mRNP proteins, their function might be simply to protect mRNAs from nucleases or to saturate nonspecific binding sites, thereby preventing the diversion of essential translation factors , some of which are also mRNA-binding proteins (Kozak 1 992). Theoretically, translation could be repressed by RNAs as well as by proteins, but there is no compelling evidence in eukaryotes for regulatory RNAs that target specific mRNAs. When one claim along those lines was followed up, the inhibitor turned out to be heparin in the RNA preparation (Johansson et al 1991). Activator Proteins Positive regulation of translation by mRNA-binding proteins is a rare phenomenon. The most compelling example from prokaryotic systems in volves the Com protein of bacteriophage Mu, which binds just upstream from the mom ribosome-binding site, thereby destabilizing a stem-and-Ioop structure that sequesters the mom initiator codon in the absence of Com (Hattman et al 1 99 1 ; Wulczyn & Kahmann 1 99 1 ) . The mom gene encodes a Mu-specific DNA modification activity that protects phage DNA from host restriction endonucleases. Since unscheduled production of the modifying activity would be lethal, the dependence of mom translation on prior synthesis of Com protein is a crucial regulatory circuit. In eukaryotes there are no unambiguous examples of mRNA-binding proteins that directly activate translation . While it is not impossible to envision how such proteins might work, the scanning mechanism of initiation would seem to impose severe constraints. For the hypothetical protein shown in Figure 2 to function as an activator and not as a repressor of translation , the protein that binds to the mRNA in step b would have to be displaced in step c. Hairpin structure B-A', the formation of which in step b exposes the 5' -end of the mRNA to ribosomes, would also have to be disrupted in step c. By analogy with the way ribosomes handle synthetic , hairpin-containing rnRNAs (Kozak 1 986a, 1 989b), the processivity of the scanning 40S ribosome should disrupt structure B-A' , provided that it is not too stable or too near the 5'-terminus. By analogy with IRE-BP (Goossen & Hentze 1 992), the hypothetical activator protein might be displaced by the scanning 40S ribosome provided that the protein-binding site is far enough from the 5' -end to permit the 40S ribosome to enter. These constraints might cause proteins that directly activate translation by binding to the mRNA template to be rare or nonexistent in eukaryotes . Some intriguing mRNA-specific proteins that do promote translation in eukaryotes, probably by an indirect mechanism, have been described, however.
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A hypothetical mechanism whereby an mRNA-binding protein might activate translation in mammalian systems. (a) In the absence of a regulatory protein, the 5' -end of the mRNA would be sequestered by base-pairing (structure A-B), and therefore the 40S ribosomal
subunit could not enter. (b) The activator protein (triangle) is postulated to stabilize an alternative stem-and-loop structure (B-A'), which opens the 5' -end of the mRNA. The transition from step
b to step
c
invokes the processivity of the scanning 40S ribosome/factor complex to displace the
activator protein and disrupt the stem-loop structure.
One is the l OO-kd nonstructural L4 protein of adenovirus that is required for efficient translation of viral late mRNAs (Hayes et al 1 990). A special trans-acting protein required for the translation of caulimovirus mRNAs is discussed in the section on reinitiation. The Rev and Tat proteins of human immunodeficiency virus modulate several steps in gene expression, but it is unclear whether translation is one of the affected steps. The stimulation of Gag protein synthesis by Rev is brought about in part by stabilization of gag/pol mRNA (Schwartz et al 1992) and by effects of Rev on the intracellular routing of gag/pol and vpu/env mRNAs. Thus when the localization of these mRNAs was examined in some experimental systems by in situ hybridization, most of the transcripts were found in the cytoplasm in the presence of Rev protein, while in the absence of Rev, the mRNAs accumulated in and around the nucleus (D' Agostino et al 1992; Lawrence et al 199 1 ) . In some other experimental systems, however, in which the absence of Rev protein did not restrict mRNAs to the nucleus, the transcripts that entered the cytoplasm appeared unable to engage ribosomes (D' Agostino et a1 1 992; Lawrence et al 1 99 1 ; Arrigo & Chen 1 99 1) . While it is possible in the latter instances to argue that Rev directly promotes translation , the effect is more likely to be indirect since Rev has no detectable effects on translation in vitro, and the site in mRNA to which Rev binds occurs downstream from the affected coding sequence. The same function of Rev that affects mRNA routing between the nucleus and cytoplasm (see above) might affect transcript localization in the cytosol, and proper compartmentalization within the cytoplasm might be a prerequisite for translation. Stimulation of translation by Tat protein also appears to be indirect, since the facilitating interaction of Tat with its target RNA must occur before the mRNA exits from the nucleus (Braddock et al 1 990, 1 99 1). Despite considerable speculation and searching, no messenger-specific
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translation initiation factors have yet been identified. There is one mRNA specific elongation factor that is involved in selenoprotein synthesis in bacteria (Forchhammer et aI1990), and there are nuclear-encoded factors that promote the translation of specific mRNAs in mitochondria (McMullin et al 1990) and chloroplasts (Danon & Mayfield 1991) in ways yet to be determined. Mammalian cells contain proteins that bind to the 5' -end of one or another viral RNA (reviewed by Kozak 1992), but mere binding to mRNA does not indicate that a protein is involved in translation; the 5'-ends of viral RNAs also participate in replication, packaging, and other activities that might be mediated by mRNA-binding proteins. The regulation of translation thus differs profoundly from the regulation of transcription, where gene-specific initiation factors abound. Instead, translational control bears a resemblance to alternative splicing, where regulation may be achieved by modulating the level of general splicing factors and by splice site-specific repressor proteins (Maniatis 1991).
REINITIATION The ability of eukaryotic ribosomes to reinitiate translation was first deduced from manipulations carried out with SV40-based expression vectors. Several investigators noticed that the inhibitory effect of an adventitious AUG codon, introduced upstream from the major open reading frame (ORF), was mitigated upon introducing a terminator codon between the upstream AUG codon and the start of the major ORF (Kozak 1984; Liu et al 1984; Peabody et al 1986). The simplest interpretation was to postulate that after an 80S ribosome has translated the upstream ORF and the 60S subunit has dissociated, the 40S ribosomal subunit holds on to the mRNA, reSUmes scanning, and is able to reinitiate translation from another AUG codon downstream. The efficiency of reinitiation was found to increase as the distance between the upstream and downstream ORFs increased (Kozak 1987b)-a surprising result since rein itiation in prokaryotic systems is usually more efficient when the 5' and 3' ORFs overlap. To rationalize the facilitating effect of intercistronic distance on reinitiation in eukaryotes, it was suggested that when the 40S ribosomal subunit resumes scanning at the end of ORF-l, it might at first be unable to recognize a downstream AUG codon, inasmuch as the 40S ribosome would lack Met-tRNAFet . Increasing the intercistronic distance would provide more time for the 40S subunit to acquire Met-tRNA and thus become competent to reinitiate (Kozak 1987b). That idea underlies the regulated reinitiation mechanism recently elucidated for the yeast GCN4 gene. The GCN4 gene of Saccharomyces cerevisiae encodes a transcription factor that activates the expression of 30-40 genes involved in amino acid biosynthesis. Expression of GCN4 itself is regulated at the level of translation: the mRNA is present constitutively, but GCN4 protein is produced only when
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cells are starved for amino acids . Because there are four small ORFs between the 5' -end of the mRNA and the start codon for GCN4, translation of GCN4 must involve a reinitiation mechanism2• An outline of the way in which the intracellular concentration of eIF-2'GTP' Met-tRNA regulates reinitiation and, in tum, is regulated by general amino acid availability has emerged from genetic and biochemical probing of this interesting system. In short, the accumulation of uncharged tRNAs under conditions of amino acid starvation leads to activation of a protein kinase that phosphorylates and thus inactivates a portion of the eIF-2 molecules (Dever et al 1 992) . Since eIF-2 is the factor that escorts Met-tRNAFet onto ribosomes, depletion of eIF-2 causes an increase in the time required for 40S ribosomal subunits to acquire Met-tRNA and thus become competent to reinitiate. As explained in Figure 3, the slower acquisition of competence under starvation conditions causes some 40S subunits to bypass one of the small upstream ORFs and, as a result, ribosomes gain access to the GCN4 start site. The complicated interplay of upstream ORFs and trans-acting regulatory proteins, one of which is the aforementioned eIF-2 kinase, is described in greater detail by Hinnebusch ( 1990). Some aspects of GCN4 regulation have been reproduced in a cell-free translation system (Krupitza & Thireos 1990). Another yeast mRNA that harbors an essential, small upstream ORF derives from the CPA1 gene (Werner et al 1 990). Here the regulatory mechanism differs significantly from GCN4 in that it is a 25-amino acid peptide encoded within the CPA1 leader sequence , and not simply the structure of the 5' -end of the mRNA that confers translational regulation. The exact mechanism by which the leader peptide represses CPA1 translation is not yet known. Among plant and animal viruses there are several examples of transcripts with small upstream ORFs that employ a reinitiation mechanism of translation (Hackett et al 1 986; Khalili et al 1 987; Schwartz et al 1 990; Sedman et al 1 989; Tollefson et al 1990), but in these cases reinitiation occurs in a constitutive rather than the regulated fashion described for yeast. The major consequence seems to be a reduction of translational efficiency. The poly merase gene of hepatitis B virus is noteworthy in that reinitiation near the 5' -end of the core/pol transcript may set up a shunt that delivers ribosomes to the internal start site for pol translation [(Ou et al 1 990; c. Shih, personal communication); a hypothetical reinitation shunt similar to that proposed for hepatitis B virus is illustrated and explained elsewhere (Figure 2 in Kozak 1 986c) ] . The most compelling example of reinitiation in multicellular 2
In mammalian systems there are two ways to skirt the first AUG rule: ribosomes can reach a downstream ORF by reinitiation or by "leaky scanning." The latter process occurs when the first AUG codon lies in an unfavorable context for initiation (Kozak 1 986b, 1 99 1 a). In yeast systems, however, there is little evidence for context effects on initiation (Cigan et al 1 988b) and, therefore, reinitiation is the only recognized mechanism for utilizing a downstream AUG codon.
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(a) Under nonstarvation conditions GCN4 is not translated.
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Annu. Rev. Cell Bioi. 1992. 8:197-225 Copyright © 1992 by Annual Reviews Inc. All rights reserved
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REGULATION OF TRANSLATION IN
Annu. Rev. Cell. Biol. 1992.8:197-225. Downloaded from www.annualreviews.org by ILLINOIS STATE UNIVERSITY on 11/17/12. For personal use only.
EUKARYOTIC SYSTEMS Marilyn Kozak Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854 KEY WORDS: protein synthesis, reinitiation, mRNA-binding proteins, mRNA structure, initia tion factors
CONTENTS 1 97
INTRODUCTION AN OVERVIEW OF TRANSLATIONAL REGULATION IN PROKARYOTIC AND EUKARYOTIC SYSTEMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
mRNA-SPECIFIC REGULATORY PROTEINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Repressor Proteins ..... .. . ......................... ...... ...... ... Activator Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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GLOBAL REGULATION OF TRANSLATION IN MAMMALIAN CELLS. . .. . . . . Phosphorylation of General Components of the Translational Machinery . . . . . . . . Targeting the Consequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 05
SELECTED ASPECTS OF TRANSLATION IN VIRUS-INFECTED CELLS .. . . . . . The Host ShutoffPhenomenon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Poliovirus Translation .............................................
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REINITIATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
FUTURE PROSPECTS .
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INTRODUCTION The striking differences between prokaryotes and eukaryotes in cell structure, in the mechanism of initiation of translation, and in the structure of messenger RNAs dictate strikingly different approaches to regulating translation. The first section of this review gives a thumbnail comparison of translational regulation in prokaryotic and eukaryotic systems. The remaining sections expand the discussion of regulatory mechanisms that operate during the initiation phase of protein synthesis in eukaryotes . 197 0743-4634/92/1115-0197$02.00
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In the field of eukaryotic gene expression there are at the moment three outstanding examples of translational regulation: (a) inhibition of ferritin translation by an iron-responsive mRNA-binding protein (IRE-BP); (b) modulation of translation via eukaryotic initiation factor-2 (eIF-2); and (c) induction of yeast GCN4 translation in response to amino acid starvation, a process that involves regulated reinitiation of translation and is a special case of regulation by eIF-2. These three paradigms, which have been elucidated in considerable molecular detail, are described herein at length , and some other emerging examples of translational control are considered briefly.
AN OVERVIEW OF TRANSLATIONAL REGULATION IN PROKARYOTIC AND EUKARYOTIC SYSTEMS A key difference between prokaryotes and eukaryotes concerns the basic mechanism whereby ribosomes engage the mRNA. Whereas prokaryotic ribosomes enter directly at the AUG initiator codon or at the nearby Shine-Dalgamo site (Calogero et al 1 988), eukaryotic ribosomes apparently enter at the capped 5' -end of the mRNA and advance to the AUG codon by linear scanning (Kozak 1989a). The prevalence of polycistronic mRNAs in prokaryotes vs monocistronic mRNAs in eukaryotes follows from that basic difference in the mechanism of initiation. Whereas the initial contact between bacterial ribosomes and mRNA requires no initiation factors or cofactors (Calogero et al 1988) , ATP and a considerable number of protein factors are needed for eukaryotic ribosomes to engage the mRNA (Hershey 1 99 1) . A further difference is that in prokaryotes the small ribosomal subunit can engage mRNA at the Shine-Dalgarno site before binding initiator Met-tRNA (Hartz et al 1 99 1 ) , whereas in eukaryotes, the small ribosomal subunit binds stably to mRNA only after initiator Met-tRNA has bound. Nevertheless, in both systems , actual recognition of the AUG initiator codon is accomplished primarily by base-pairing with the complementary anticodon in Met-tRNA , as evidenced b y the ability to force initiation from a non-AUG codon when the anticodon sequence is experimentally changed (Cigan et al 1 988a; Varshney & RajBhandary 1 990) . Other basic aspects of the initiation mechanisms in prokaryotic and eukaryotic organisms have been reviewed previously (Gold 1 988; Hershey 1 99 1 ; Kozak 1 983) . Between prokaryotes and eukaryotes there are more differences than similarities in the mechanisms whereby translation is regulated. In prokaryotic organisms (Gold 1988; McCarthy & Gualerzi 1990) , two mechanisms are widespread: (a) repression of translation by mRNA-binding proteins , of which at least 16 examples have been described (Andrake et a1 1 988; Dolan & Oliver 199 1 ; Kozak 1 988a) , and (b) regulation via switches in mRNA conformation that expose a previously sequestered ribosome-binding site. An excellent
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review by de Smit & van Duin (1990) illustrates some of the mRNA structures involved in this type of regulation. The conformational switches may be brought about by the binding of an activator protein (Altuvia et al 1991; Hattman et al 1991; Wu1czyn & Kahmann 1991), or by translation of the upstream portion of the mRNA (Asano et al 1 99 1 ; Berkhout & van Duin 1 985; Dick & Matzura 1 990; Mayford & Weisblum 1 989; Rogers et a1 1 99 1 ), or by endonuc1eolytic processing of the transcript (Dunn & Studier 1975), or by a switch in the start site for transcription (Ambulos et al 1 99 1 ; MacDonald et al 1 984; Schulz & Reznikoff 1 99 1 ) . In eukaryotes, on the other hand, there is only one documented example of a translational repressor protein (the IRE-BP mentioned above), no proven translational activator proteins, and no known regulation by switches in mRNA conformation. To the extent that activation of translation by a change in mRNA conformation might occur in eukaryotes, one would predict that it should involve exposure of the capped 5' -end of the mRNA-the apparent entry site for 40S ribosomes-rather than exposure of the AUG initiator codon. Regulation of translation by small naturally occurring antisense RNAs has been documented occasionally in prokaryotes (Citron & Schuster 1 990; Inouye 1988; Ma & Simons 1990; Wagner et al 1987), but not thus far in eukaryotes . [Anti-sense RNAs that have been detected rarely in eukaryotic cells apparently operate at a pretranslational level (Kimelman & Kirschner 1989; Skeiky & Iatrou 1990; Tosic et al 1990).] In prokaryotes, reinitiation may occur during the coupled translation of cotranscribed genes, but reinitia tion in prokaryotes apparently is not regulated; i.e. it does not become more or less efficient in response to changes in the intracellular environment. Thus regulated reinitiation, as discussed below for the yeast GCN4 gene, is unique to eukaryotic systems. While we can count one certain example of a translational repressor protein in eukaryotes and one proven instance of regulated reinitiation, both described below, the number of eukaryotic genes for which specific translational regulatory mechanisms have been defined is much, much smaller than the number of prokaryotic genes that can be so counted. I suspect the explanation is not that translational regulation is less common in eukaryotes, but that it often takes forms that are harder to recognize. For example, modifications of general components of the translational machinery, which are virtually unheard of in prokaryotes, are widespread in eUkaryotic cells; and, at least in the case of eIF-2, the modification impairs the ability of the factor to support protein synthesis. If the resulting global throttling of the translational capacity constitutes a selective regulatory device, however, it would have to be targeted somehow--if not to specific mRNAs, then to groups of mRNAs. Some speculations about how that might occur are offered below. The possibility that translational control, especially in developing systems , might involve
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compartmentalization of mRNAs and/or components of the translational machinery is also discussed, although the available data are inadequate to resolve the issue. The possibility of regulation at the level of polypeptide chain elongation has been considered elsewhere (Ryazanov et al 1 99 1 ) . The mechanisms mentioned above effect switches i n translation, which i s the way translational regulation i s usually defined. But the definition might be broadened to include a substantial number of mRNAs that have, built into their invariant structures, features that force the standard translational ma chinery to perform in nonstandard ways-- initiating translation from two sites, for example, or shifting to a different reading frame during the course of elongation, or translating through a stop codon. Those aspects of translational control are discussed in other reviews (Atkins et al 1 990; Kozak 1 99 1 a).
mRNA-SPECIFIC REGULPJORY PROTEINS Repressor Proteins In mammalian cells, synthesis of the iron-sequestering protein ferritin is regulated by iron availability (Klausner & Harford 1 989). The regulation is accomplished by a �90-kd protein that inhibits translation by binding near the 5'-end of ferritin mRNA (Figure la). The target site for the repressor, a conserved stem-and-loop structure depicted in Figure Ie and d, is called appropriately the iron-response element (IRE), and the repressor protein is the IRE-binding protein (IRE-BP). A recently obtained full-length cDNA clone for murine IRE-BP reveals that the repressor is related to mitochondrial aconitase , and enzymatic tests reveal that IRE-BP itself has aconitase activity (Kaptain et al 1 99 1 ) . The fact that aconitase contains a labile [Fe-S] complex suggests a likely mechanism whereby IRE-BP senses iron levels. As illustrated in Figure 1 , the reversible binding of IRE-BP to mRNA is thought to reflect reversible oxidation of a critical sulfhydryl group: when iron is scarce, the reduced [3Fe-4S] form of the protein binds the IRE; when iron is abundant, the oxidized [4Fe-4S] protein dissociates from the IRE (Haile et al 1 989; Rouault et al 1991; Constable et al 1992). The identification of IRE-BP as a cytosolic aconitase offers interesting insight into how a eukaryotic repressor protein evolves. One is reminded of the situation in prokaryotes, where virtually every translational repressor protein has a primary function other than regulating translation (Kozak 1 988a)! The effects of IRE-BP on ferritin translation were deduced from model reactions between IRE-BP and mRNA fragments (Rouault et al 1 98 8; Barton et al 1 990; Constable et al 1 992), from the effects of IRE mutations on translation in vivo (Caughman et al 1 988), and from direct studies of translation in vitro (Brown et al 1 989). Qualitatively those results support the model in Figure la, but the range of induction in some test systems is
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(a) Regulated Translation ot Ferritin mRNA
(b) Regulated Stability 01 Transterrin Receptor mRNA
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Figure 1
Post-transcriptional regulation of two genes involved in the uptake and detoxification of iron in mammalian cells. Regulation is accomplished by the interaction of a protein, IRE-BP (shown as a triangle), with a specific target sequence IRE (shown as a hairpin in the mRNA). The total amount of IRE-BP in cells is invariant, but the affinity of the protein for its target site varies with the iron status ofthe cell, as explained in the text. (a) Translation offerritin, a protein that sequesters excess iron, is inhibited by the binding of IRE-BP to ferritin mRNA. (b) IRE-BP binds to and stabilizes the mRNA that encodes transferrin receptor ( TfR) , a protein that promotes the uptake of iron into cells.The 3'-noncoding sequence of TfR mRNA contains five IRE motifs, only one of which is shown. The presumptive target sites for ribonuclease(s) near the 3' -end of TtR mRNA have not yet been mapped precisely. The actual sequence of one IRE (c) is shown along with a consensus IRE structure (d) derived from phylogenetic and other data (Hentze et al 1988; Leibold et al 1 990).
considerably less than what has been observed with endogenous ferritin genes, which suggests that control of ferritin synthesis might occur at multiple levels (Mattia et al 1 989; Goessling et al 1 992). While translation can be inhibited by base-paired structures per se (Kozak 1 986a, 1 989b), the particular stem-loop structure that constitutes the IRE is not very stable, which probably explains why it does not inhibit translation on its own. The binding of IRE-BP may stabilize the base-paired component of the IRE (Harrell et al 1 99 1 ) . In ferritin mRNAs the IRE is located typically about 30 nt from the cap \ and this 5' proximal location is very important. In a telling experiment, repression was lost when the IRE was repositioned 67 nt from the 5' -end of a test transcript (Goossen & Hentze 1 992). The simplest interpretation is that a 40S ribosomal subunit, once bound to the 5' -end of the mRNA, can displace proteins as it advances, much as the scanning 40S ribosome has been shown to melt certain base-paired structures (Kozak 1 986a, 1 989b). Given the apparent ability of the 40S ribosomal subunit to clear a path as it advances, IThe IRE is often part of an extended stem that actually begins mRNA (Wang
et al
1990).
-10 nt from the 5'-end of the
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an mRNA-binding protein (or a hairpin structure) will inhibit translation only if it is close enough to the 5 ' -end to prevent the initial binding of the 40S ribosome. It is intriguing that the same protein that down-regulates ferritin synthesis when iron is scarce has been found to up-regulate production of the transferrin receptor (TfR) protein, as illustrated in Figure lb and described more completely by Klausner & Harford ( 1 989). The opposite effects of iron deprivation on ferritin and TtR synthesis reflect the location of the IRE in each mRNA: masking by IRE-BP of the 5' -end of the mRNA understandably inhibits translation of ferritin, while masking of the 3 ' -noncoding end of the mRNA , which is irrelevant to ribosomes but is often the target of ribonucleases (Peltz et al 199 1 ) , increases the stability of TtR mRNA. Recent data suggest that IRE-BP also regulates the translation of erythroid 5' -aminolevulinate synthase, the first enzyme of the heme biosynthetic pathway (Cox et a1 1991; Dandekar et al 1 99 1 ). Thus a single remarkable protein controls both iron homeostasis and a key metabolic pathway for iron utilization. The search for other eUkaryotic genes controlled by translational repressor proteins has produced no compelling results as yet. A few candidates merit watching (Kaspar et al 1992; Lee et al 1991; Schafer et al 1990), but the evidence adduced so far is barely suggestive. The mere demonstration that one or another protein binds to an mRNA leader sequence permits no inferences about effects of the protein on translation. "Masking proteins" are a traditional explanation for some aspects of translational regulation in developing systems (Richter 1 99 1 ) , but the cis-acting elements underlying such control are often at the wrong end (i.e. the 3' rather than the 5'- end) of the mRNA, and no one has yet shown that the mRNA-binding proteins have the predicted effects on translation. The key experiments would be to show that (a) with native ribonucleoproteins (mRNPs) as templates, the pattern of translation in vitro reproduces the restricted pattern observed in vivo; (b) extraction of proteins from mRNPs specifically activates translation of the mRNAs that were restricted in the preceding step; and (c) re-addition of a purified protein fraction inhibits translation of the specific target mRNAs. It is the last step that has failed in every attempt to test the masked mRNA hypothesis (Grainger & Winkler 1987; Standart et aI 1 990). Thus while there is overwhelming evidence that certain mRNAs are present in oocytes prior to the onset of their translation, the evidence implicating soluble translational repressor proteins is not overwhelming. The fact that mRNAs differ in the extent to which they accumulate in mRNPs has been cited as evidence that RNP proteins recognize specific mRNAs (Kelso-Winemiller & Winkler 1991 ), but an alternative interpretation is that mRNAs that cannot engage ribosomes (e.g. due to secondary structure near the 5' -end) are left exposed and therefore pick up RNP proteins. In other
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words, accumulation in mRNPs might be the passive fate of untranslated mRNAs rather than a mechanism that actively restricts translation. In view of the ubiquity of mRNP proteins, their function might be simply to protect mRNAs from nucleases or to saturate nonspecific binding sites, thereby preventing the diversion of essential translation factors , some of which are also mRNA-binding proteins (Kozak 1 992). Theoretically, translation could be repressed by RNAs as well as by proteins, but there is no compelling evidence in eukaryotes for regulatory RNAs that target specific mRNAs. When one claim along those lines was followed up, the inhibitor turned out to be heparin in the RNA preparation (Johansson et al 1991). Activator Proteins Positive regulation of translation by mRNA-binding proteins is a rare phenomenon. The most compelling example from prokaryotic systems in volves the Com protein of bacteriophage Mu, which binds just upstream from the mom ribosome-binding site, thereby destabilizing a stem-and-Ioop structure that sequesters the mom initiator codon in the absence of Com (Hattman et al 1 99 1 ; Wulczyn & Kahmann 1 99 1 ) . The mom gene encodes a Mu-specific DNA modification activity that protects phage DNA from host restriction endonucleases. Since unscheduled production of the modifying activity would be lethal, the dependence of mom translation on prior synthesis of Com protein is a crucial regulatory circuit. In eukaryotes there are no unambiguous examples of mRNA-binding proteins that directly activate translation . While it is not impossible to envision how such proteins might work, the scanning mechanism of initiation would seem to impose severe constraints. For the hypothetical protein shown in Figure 2 to function as an activator and not as a repressor of translation , the protein that binds to the mRNA in step b would have to be displaced in step c. Hairpin structure B-A', the formation of which in step b exposes the 5' -end of the mRNA to ribosomes, would also have to be disrupted in step c. By analogy with the way ribosomes handle synthetic , hairpin-containing rnRNAs (Kozak 1 986a, 1 989b), the processivity of the scanning 40S ribosome should disrupt structure B-A' , provided that it is not too stable or too near the 5'-terminus. By analogy with IRE-BP (Goossen & Hentze 1 992), the hypothetical activator protein might be displaced by the scanning 40S ribosome provided that the protein-binding site is far enough from the 5' -end to permit the 40S ribosome to enter. These constraints might cause proteins that directly activate translation by binding to the mRNA template to be rare or nonexistent in eukaryotes . Some intriguing mRNA-specific proteins that do promote translation in eukaryotes, probably by an indirect mechanism, have been described, however.
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A hypothetical mechanism whereby an mRNA-binding protein might activate translation in mammalian systems. (a) In the absence of a regulatory protein, the 5' -end of the mRNA would be sequestered by base-pairing (structure A-B), and therefore the 40S ribosomal
subunit could not enter. (b) The activator protein (triangle) is postulated to stabilize an alternative stem-and-loop structure (B-A'), which opens the 5' -end of the mRNA. The transition from step
b to step
c
invokes the processivity of the scanning 40S ribosome/factor complex to displace the
activator protein and disrupt the stem-loop structure.
One is the l OO-kd nonstructural L4 protein of adenovirus that is required for efficient translation of viral late mRNAs (Hayes et al 1 990). A special trans-acting protein required for the translation of caulimovirus mRNAs is discussed in the section on reinitiation. The Rev and Tat proteins of human immunodeficiency virus modulate several steps in gene expression, but it is unclear whether translation is one of the affected steps. The stimulation of Gag protein synthesis by Rev is brought about in part by stabilization of gag/pol mRNA (Schwartz et al 1992) and by effects of Rev on the intracellular routing of gag/pol and vpu/env mRNAs. Thus when the localization of these mRNAs was examined in some experimental systems by in situ hybridization, most of the transcripts were found in the cytoplasm in the presence of Rev protein, while in the absence of Rev, the mRNAs accumulated in and around the nucleus (D' Agostino et al 1992; Lawrence et al 199 1 ) . In some other experimental systems, however, in which the absence of Rev protein did not restrict mRNAs to the nucleus, the transcripts that entered the cytoplasm appeared unable to engage ribosomes (D' Agostino et a1 1 992; Lawrence et al 1 99 1 ; Arrigo & Chen 1 99 1) . While it is possible in the latter instances to argue that Rev directly promotes translation , the effect is more likely to be indirect since Rev has no detectable effects on translation in vitro, and the site in mRNA to which Rev binds occurs downstream from the affected coding sequence. The same function of Rev that affects mRNA routing between the nucleus and cytoplasm (see above) might affect transcript localization in the cytosol, and proper compartmentalization within the cytoplasm might be a prerequisite for translation. Stimulation of translation by Tat protein also appears to be indirect, since the facilitating interaction of Tat with its target RNA must occur before the mRNA exits from the nucleus (Braddock et al 1 990, 1 99 1). Despite considerable speculation and searching, no messenger-specific
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translation initiation factors have yet been identified. There is one mRNA specific elongation factor that is involved in selenoprotein synthesis in bacteria (Forchhammer et aI1990), and there are nuclear-encoded factors that promote the translation of specific mRNAs in mitochondria (McMullin et al 1990) and chloroplasts (Danon & Mayfield 1991) in ways yet to be determined. Mammalian cells contain proteins that bind to the 5' -end of one or another viral RNA (reviewed by Kozak 1992), but mere binding to mRNA does not indicate that a protein is involved in translation; the 5'-ends of viral RNAs also participate in replication, packaging, and other activities that might be mediated by mRNA-binding proteins. The regulation of translation thus differs profoundly from the regulation of transcription, where gene-specific initiation factors abound. Instead, translational control bears a resemblance to alternative splicing, where regulation may be achieved by modulating the level of general splicing factors and by splice site-specific repressor proteins (Maniatis 1991).
REINITIATION The ability of eukaryotic ribosomes to reinitiate translation was first deduced from manipulations carried out with SV40-based expression vectors. Several investigators noticed that the inhibitory effect of an adventitious AUG codon, introduced upstream from the major open reading frame (ORF), was mitigated upon introducing a terminator codon between the upstream AUG codon and the start of the major ORF (Kozak 1984; Liu et al 1984; Peabody et al 1986). The simplest interpretation was to postulate that after an 80S ribosome has translated the upstream ORF and the 60S subunit has dissociated, the 40S ribosomal subunit holds on to the mRNA, reSUmes scanning, and is able to reinitiate translation from another AUG codon downstream. The efficiency of reinitiation was found to increase as the distance between the upstream and downstream ORFs increased (Kozak 1987b)-a surprising result since rein itiation in prokaryotic systems is usually more efficient when the 5' and 3' ORFs overlap. To rationalize the facilitating effect of intercistronic distance on reinitiation in eukaryotes, it was suggested that when the 40S ribosomal subunit resumes scanning at the end of ORF-l, it might at first be unable to recognize a downstream AUG codon, inasmuch as the 40S ribosome would lack Met-tRNAFet . Increasing the intercistronic distance would provide more time for the 40S subunit to acquire Met-tRNA and thus become competent to reinitiate (Kozak 1987b). That idea underlies the regulated reinitiation mechanism recently elucidated for the yeast GCN4 gene. The GCN4 gene of Saccharomyces cerevisiae encodes a transcription factor that activates the expression of 30-40 genes involved in amino acid biosynthesis. Expression of GCN4 itself is regulated at the level of translation: the mRNA is present constitutively, but GCN4 protein is produced only when
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cells are starved for amino acids . Because there are four small ORFs between the 5' -end of the mRNA and the start codon for GCN4, translation of GCN4 must involve a reinitiation mechanism2• An outline of the way in which the intracellular concentration of eIF-2'GTP' Met-tRNA regulates reinitiation and, in tum, is regulated by general amino acid availability has emerged from genetic and biochemical probing of this interesting system. In short, the accumulation of uncharged tRNAs under conditions of amino acid starvation leads to activation of a protein kinase that phosphorylates and thus inactivates a portion of the eIF-2 molecules (Dever et al 1 992) . Since eIF-2 is the factor that escorts Met-tRNAFet onto ribosomes, depletion of eIF-2 causes an increase in the time required for 40S ribosomal subunits to acquire Met-tRNA and thus become competent to reinitiate. As explained in Figure 3, the slower acquisition of competence under starvation conditions causes some 40S subunits to bypass one of the small upstream ORFs and, as a result, ribosomes gain access to the GCN4 start site. The complicated interplay of upstream ORFs and trans-acting regulatory proteins, one of which is the aforementioned eIF-2 kinase, is described in greater detail by Hinnebusch ( 1990). Some aspects of GCN4 regulation have been reproduced in a cell-free translation system (Krupitza & Thireos 1990). Another yeast mRNA that harbors an essential, small upstream ORF derives from the CPA1 gene (Werner et al 1 990). Here the regulatory mechanism differs significantly from GCN4 in that it is a 25-amino acid peptide encoded within the CPA1 leader sequence , and not simply the structure of the 5' -end of the mRNA that confers translational regulation. The exact mechanism by which the leader peptide represses CPA1 translation is not yet known. Among plant and animal viruses there are several examples of transcripts with small upstream ORFs that employ a reinitiation mechanism of translation (Hackett et al 1 986; Khalili et al 1 987; Schwartz et al 1 990; Sedman et al 1 989; Tollefson et al 1990), but in these cases reinitiation occurs in a constitutive rather than the regulated fashion described for yeast. The major consequence seems to be a reduction of translational efficiency. The poly merase gene of hepatitis B virus is noteworthy in that reinitiation near the 5' -end of the core/pol transcript may set up a shunt that delivers ribosomes to the internal start site for pol translation [(Ou et al 1 990; c. Shih, personal communication); a hypothetical reinitation shunt similar to that proposed for hepatitis B virus is illustrated and explained elsewhere (Figure 2 in Kozak 1 986c) ] . The most compelling example of reinitiation in multicellular 2
In mammalian systems there are two ways to skirt the first AUG rule: ribosomes can reach a downstream ORF by reinitiation or by "leaky scanning." The latter process occurs when the first AUG codon lies in an unfavorable context for initiation (Kozak 1 986b, 1 99 1 a). In yeast systems, however, there is little evidence for context effects on initiation (Cigan et al 1 988b) and, therefore, reinitiation is the only recognized mechanism for utilizing a downstream AUG codon.
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(a) Under nonstarvation conditions GCN4 is not translated.
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