The scanning model for translation: an update.

The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained.

T HE scanning mechanism for initiation of translation in eukaryotes was proposed 10 years ago (112). Supporting evidence has accumulated at a slower rate than one might have wished, but a trickle sustained over 10 yr forms a decent-sized pond. Some remarkable experiments now being carried out in yeasts are yielding important new insights about scanning and other aspects of initiation, and the power of the yeast system promises additional breakthroughs in the coming years. Thus, it seems a good time to review what we've learned so far about how eukaryotic ribosomes select a particular AUG codon as the start site for translation.

Evidence from Higher Eukaryotes
The scanning model states that the 40S ribosomal subunit (carrying Met-tRNA~ me, and various initiation factors) binds initially at the 5'-end of mRNA and then migrates, stopping at the first AUG codon in a favorable context for initiating translation. The model thus posits that both position (proximity to the 5'-end) and context contribute to selection of the initiation site. The simplest evidence of the importance of position is that the "first-AUG rule" holds for some 90-95 % of the hundreds of vertebrate mRNA sequences that have been analyzed (127). A detailed discussion of position effects, along with an explanation for the 5-10% deviation from the first-AUG rule, will follow after a brief introduction to the context requirements for initiation.
From a recent survey of 699 vertebrate mRNAs (127), GCCGCC~CCAUGG emerged as the consensus sequence for initiation in higher eukaryotes, confirming and extending previously noted trends (117,120). Site-directed mutagenesis experiments in which the expression of a cloned preproinsulin gene was monitored in transfected COS cells have confirmed the importance of G +4 as well as each of the consensus nucleotides from position -1 through -6 (122, 126)'. The importance of context was demonstrated by targeting mutations to the vicinity of the AUG initiator codon for preproinsulin as well as by targeting an upstream, out-of-1. Numbering begins with the A of the AUG codon as position +1; nucleotides 5' to that site are assigned negative numbers. frame AUG codon; in the latter case the inhibitory effect of the upstream AUG codon (i.e., its ability to block the initiation of preproinsulin from a downstream site) increased as the surrounding nucleotides increasingly resembled the consensus sequence. The importance of a purine in position -3 was confirmed by the discovery of a type of thalassemia in which a change in sequence, from CACCAUG to CCCCAUG, drastically impairs initiation of translation of a-globin (167). Experiments confirming (66, 92,192,248,255) and exploiting the effects of context on initiation (169,181,222,263) 2 have recently been reported from other systems. A purine (usually A) in position -3 is the most highly conserved nucleotide in all eukaryotic mRNAs, including those of vertebrates, plants (81), and fungi (188); and a mutation in that position affects translation more profoundly than a point mutation anywhere else (122). Indeed, as long as there is a pufine in position -3, deviations from the rest of the consensus sequence only marginally impair initiation. In the absence of a purine in position -3, however, G ÷4 is essential for efficient translation (122) and the contributions of other nearby nucleotides can be detected (126). For practical purposes, an initiator codon can usually be designated "strong" or "weak" by considering only positions -3 and +4; I shall follow that convention in the rest of this paper.
The scanning model predicts that proximity to the 5'-end determines which AUG codon (in a good context) actually functions as the initiator codon. The nearly simultaneous discoveries of the mTG cap (232) and of "silent" 3'-cistrons in many viral mRNAs (243) constituted the first strong evidence that eukaryotic ribosomes are somehow restricted to initiating near the 5'-end. Some of the early in vitro evidence for silent Y-cistrons has recently been confirmed in vivo (70,281). In lieu of a scanning mechanism to explain the 5' restriction in viral mRNAs, one might have postulated that the primary and/or secondary structure around a particular AUG triplet (which just happened to be 5' proximal) caused it to be the preferred initiation site. To rigorously test the importance of position, therefore, a plasmid with four identical copies of the preproinsulin "ribosome binding site" was constructed and tested (118); the outcome, exclusive use of the first site in the tandem array, strongly supports a scanning mechanism. The scanning model predicts that, upon introducing an adventitious upstream AUG codon (in a good context), initiation should shift to the upstream site. Thus, if an upstream AUG codon is in the wrong reading frame, it should, and does, depress the yield of protein from the normal site (10, 39, 70,105,144,156,198,214,282). Ira strong AUG codon is introduced upstream from, and in the same reading frame as, the normal initiator codon, the result is a stretched polypeptide with an NH2-terminal amino acid extension. This has been documented with laboratory constructs (87,118,132,148,228,244) and the same principle operates with some cellular and viral genes that use two promoters for transcription. In the latter cases, the longer transcript has an extra AUG codon (in a good context) upstream from the start of the shorter transcript; ribosomes initiate exclusively at the first AUG codon in each form of mRNA, producing "long" and "short" versions of the encoded protein that often mediate different biological functions (130). (If the Y-proximal AUG codon in the longer mRNA were in a less favorable context, both versions of the protein could be translated from a single mRNA, as described below.) Besides the wealth of evidence relating to position effects, here is some of the other evidence that supports and defines the scanning mechanism: (a) That the 40S ribosomal subunit/factor complex can migrate was deduced from the properties of complexes formed between reovirus mRNAs and wheat germ ribosomes in the presence of edeine (131). Those observations were subsequently confirmed with extracts from mammalian cells (35). (b) Other in vitro experiments reveCaled that sliding requires ATP hydrolysis and that, in the absence of ATP, 40S subunits are trapped upstream from the AUG codon, as the scanning model predicts (115). (c) The inability of eukaryotic ribosomes to bind to circular mRNAs is strong evidence against direct binding at the AUG start site (109,113). That site was identical in the linear and circular forms of each template, but only the linear form could bind to wheat germ or reticulocyte ribosomes. Control experiments showed that both linear and circular templates bound to bacterial ribosomes, emphasizing the fundamental difference between prokaryotes and eukaryotes in the mechanism of initiation (119). (d) Translation of various mRNAs is inhibited when a hairpin structure, or a DNA-RNA hybrid, or a hybrid with anti-sense RNA is introduced near the 5'-end of the mRNA in a way that does not occlude the AUG codon or the m7G cap (36, 75,123). The simplest interpretation, albeit not yet verified by footprinting, is that 40S ribosomal subunits can still bind to such mRNAs but cannot migrate beyond the duplex barrier.

Evidence from Yeasts
Yeast mRNA sequences resemble those of beasts in that translation begins at the first AUG codon in 95 % of the genes examined (29). The rare occurrence of upstream AUG codons is neither an accident, nor a problem, but a means to regulate the translation of some interesting genes (168,250,261,277). Two yeast genes have been subjected to ex-haustive genetic probing, the results of which strongly support a scanning mechanism. One set of experiments began with a CYC1 allele in which the normal AUG initiator codon had been inactivated (235). From the structure of revertants that had regained cytochrome c function, it appeared as if an AUG codon introduced anywhere within a stretch of 37 nucleotides could function in initiation. Donahue and Cigan (44) used a complementary approach to reach the same basic conclusion. They began with a specially designed HIS4 allele in which initiation at the 5'-proximal AUG codon gave a His-phenotype, while initiation from the next AUG downstream restored gene expression. That unique genetic behavior enabled them to select for mutations that reduced or abolished ribosomal recognition of the first AUG codon. The only point mutations identified in the search were alterations in the AUG codon itself, elegantly confirming the importance of "position" and suggesting that, if flanking sequences affect recognition of the AUG codon in yeast, the effects must be subtle.
When the latter question was addressed directly, by mutating nucleotides in the vicinity of the AUG codon, only modest effects (twofold or less) were found (6, 31, 277, 287). Indeed, although yeast mRNA sequences almost always have an A in position -3, the rest of the vertebrate consensus sequence is not evident (29), suggesting a different role for (or different sensitivity to) context in the two systems. Even the conserved A -3 might simply reflect the overall A richness of 5'-noncoding sequences in lower eukaryotes. The significance of the A-rich, G-deficient leader sequences on mRNAs from yeasts and other lower eukaryotes (26,48,51,104,172,208,245) is unknown, but an inevitable consequence is that such leader sequences lack extensive secondary structure, which seems to be more of an impediment to translation in yeasts (6, 31) than in higher eukaryotes (123).
Other recent experiments from Donahue's laboratory have yielded one remarkable new insight and validated one critical old assumption about the mechanism of initiation of translation. This time Donahue's group began by inactivating the AUG initiator codon in the yeast HIS4 gene. Their subsequent search for second-site suppressor mutations led them to eIF2, the protein factor that escorts Met-tRNA# el onto the ribosome. They found that a mutation in the/$ subunit of eIF2 suppressed the His-phenotype by allowing ribosomes to initiate at the first UUG codon in HIS4 mRNA (45).
Thus, eIF2 is an active participant (and the only protein factor so far implicated by genetic criteria!) in the mRNA binding-and-scanning step of initiation. The elegant followup experiment was to change the anticodon sequence from 3'-UAC-5' to 3'-UCC-5' in one of the tRNA~ m°t genes of S. cerevisiae; the mutant form of tRNA~ m¢t directed ribosomes to initiate at (the first) AGG instead of the usual AUG codon (30). This is direct proof that the initiator codon is recognized primarily by base pairing with the anticodon in Met-tRNA~m% as had always been (only) supposed. And the experiment is compelling proof of scanning.

Explicable Exceptions to the First-AUG Rule
There are a number of well-characterized viral and cellular mRNAs in which translation is not limited to the AUG codon nearest the 5'-end, but even these "exceptional" mRNAs adhere to rules that are consistent with a scanning mechanism. For example, although initiation is not restricted to the first AUG codon in the examples discussed below, initiation is at least limited to AUG codons in the vicinity of the 5'-end. In no case do eukaryotic ribosomes initiate de novo in the middle of an mRNA. In several cases in which one or more small open reading frames (ORFs) 3 precede the major ORF, the small upstream ORFs are translated (65, 76,91,101,277). The "problem 7 therefore, is not that the 5'-proximal AUG codon is missed but that it is not used exclusively.
Initiation at downstream AUG codons occurs, not haphazardly, but under three specific conditions: (a) When there are fewer than 10 nucleotides between the cap and the first AUG codon, ribosomes may initiate at the first and second AUG codons (99,199,251). This rule, deduced from the behavior of natural mRNAs, has not yet been verified by systematic experiments; it is supported, however, by some results of manipulating late leader sequences on SV-40 mRNAs (39, 72). (b) When the first AUG codon lies in an unfavorable context for initiation (i.e., when position -3 is C or U; or when position -3 is G and position +4 is not G), "leaky scanning" enables some ribosomes to reach and initiate at the second AUG codon. The hypothesis (125) that leaky scanning underlies the ability of bifunctional viral mRNAs to direct the synthesis of two separately initiated proteins is supported by the effects of mutations on the translation of two such mRNAs. Thus, Sedman and Mertz (228) probed the translation of SV-40 late 19S mRNA by introducing mutations near the 5'-end, and found that the relative yields of VP2 and VP3 (the second initiated downstream from the first) varied in accordance with the scanning rules. Mutagenesis of the Sendai virus P/C gene (where proteins P and C are translated from one mRNA in different, overlapping reading frames) also gave results consistent with leaky scanning, with the interesting twist that initiation occurs from three sites in that message: the first ACG codon functions because it lies in an excellent context (GCCACGG), but functions poorly because it is not AUG; the next AUG codon in a rather poor context (CGCAUGG) functions inefficiently; thus most 40S subunits advance to and initiate at the third start site, AAGAUGC (37, 74). Thus far, 17 viral mRNAs (reviewed in reference 124) have been shown to produce two, or rarely three, overlapping proteins by initiating at a weak upstream, as well as the next downstream, AUG codon. An important addition to the list of bifunctional mRNAs is the pX transcript of human T-cell leukemia virus type I, which directs synthesis of both p27 (from the first, weak AUG codon) and p40 (173,241). The recently determined sequences of turnip yellow mosaic virus genomic RNA (165) and one of the simian rotavirus SAIl gene segments (164) suggest that they, too, should produce two proteins by leaky scanning. The only viral mRNA known to produce two proteins from overlapping reading frames, the first of which initiates with an anomalously strong AUG codon, is the NA/NB mRNA of influenza B virus (234). The explanation in that case might involve slippage on the run of A residues flanking the first AUG codon. (c) The third condition that allows access to internal AUG codons is reinitiation. When an AUG codon upstream from the start of the major protein coding sequence lies in a favorable context, thereby precluding leaky scanning, ribosomes can still reach the downstream initiation site provided that a terminator codon occurs in-3. Abbreviation used in this paper: ORF, open reading frame. frame with the first AUG codon and upstream from the second (121,128,148). In such cases the first (invariably small) ORF is translated (65, 76,91,101,277), after which 40S ribosomal subunits apparently resume scanning and reinitiate farther downstream. (If initiation factors dissociate from the 40S subunit slowly rather than instantaneously, upon the addition of a 60S subunit and commencement of peptide bond formation, reinitiation might be possible after the translation of an upstream "minicistron, as has been observed, but not after the translation of a full-sized cistron. That as-yet-untested hypothesis would explain why 3'-cistrons in viral mRNAs are usually silent [243].) The influence of flanking sequences on AUG codon recognition follows the same hierarchy in the reinitiation mode as in the primary scanning mode (122), but not every downstream AUG codon in a good context is an efficient site for reinitiation. The efficiency of reinitiation at a downstream AUG codon steadily improves, for example, as its distance from the upstream ORF increases. Consequently, reinitiation is not limited to the first strong AUG codon after the 5'-proximal ORE (See reference 128 for evidence and further discussion of this.) Because reinitiation is usually inefficient with natural mRNAs, the presence of short upstream ORFs usually reduces translation of the downstream ORF (69,72,102,185), albeit not as severely as were there no terminator codon between the upstream AUG codon and the downstream ORE Failure to consider the contribution of leaky scanning probably explains some reports (193,262) in which the apparent requirements for reinitiation differed from what I have described here.

Some Harder Cases
The popular press (88) has recently announced that picornaviruses "break the rules" by allowing a 40S ribosomal subunit to bind directly to an internal site (somewhere upstream from the start of the major ORF) in lieu of the usual end-dependent mode of entry. The key experiments (196) involve the translation of a dicistronic transcript of the form TK-PV-CAT, where the 736-nucleotide poliovirus leader sequence (PV) separates the Y-proximal thymidine kinase gene (TK) from the T-proximal chloramphenicol acetyltransferase gene (CAT). COS cells transfected with the dicistronlc vector clearly produced some CAT protein, but that result hardly warrants the conclusion that the presence of the poliovirus leader sequence allows the efficient translation of CAT by direct internal initiation. One problem is that efficiency was claimed without having been demonstrated; i.e., it was not shown how much CAT protein was produced in vivo from the dicistronic vector relative to a monocistronic CAT transcript that bears a normal leader sequence: The most serious deficiency, however, is that the Northern blot which was offered as proof that the dicistronic transcript is the only form of CAT mRNA in transfected cells was much too faint to prove the point. When translation of dicistronic mRNAs was studied in vitro, on the other hand, the unbound mRNA pool was found to be completely degraded after only 10 rnin of incubation, which makes it hard to believe that many, if any, transcripts were intact after 60 rain, when the CAT yield was measured. In the absence of rigorous proof that dicistronic mRNAs are the only transcripts available for translation, the recent picornavirus experiments (90,196) are no different from older in vitro experiments (see below) in which the appearance of internal initiation has turned out to be an artifact. That some dicistronic mRNA was associated with polysomes (196) is not surprising, since the Y-proximal TK cistron would be functional. To prove that the 3'-proximal CAT cistron could also function, the authors should have immunoprecipitated polysomes engaged in CAT synthesis and asked whether all (or any!) of the mRNA thus selected was dicistronic. In capable hands such experiments can yield answers (8). In a different approach, Pelletier and Sonenberg (196) showed that their dicistronic transcript formed "disomes" in the presence of sparsomycin, from which they assumed, without direct evidence, that one of the two ribosomes was bound to the PV sequence in the middle of the transcript. Years ago similar ideas were entertained with other viral mRNAs that formed disomes under conditions of initiation (201); but, in every instance where the ribosome-protected sites were sequenced, both sites mapped to the 5'-noncoding sequence (2, 202,267). Thus the mere formation of disomes in sparsomycin-inhibited extracts is not evidence of a functional 3'-cistron. In view of all these shortcomings, all we yet know about the translation of poliovirus mRNA is that it occurs in the absence of the usual m7G cap and despite the burden of eight (mostly weak) upstream AUG codons (207).
Turning from viral to cellular mRNAs, the 5-10% of vertebrate mRNAs 5 that have upstream AUG codons are an interesting, nonrandom set. Many protooncogenes and growthcontrol genes produce mRNAs with upstream AUG codons (41, 68, 189,215,273,283,285) 6 which may be lost during the rearrangements that accompany activation (156 and references therein). The cDNAs that have been characterized from homeobox genes are also likely to have upstream AUG codons (24, 239), although it is not clear that those cDNAs correspond to functional mRNAs. The transcript that corresponds to one AUG-burdened homeobox cDNA, for example, is restricted to the nucleus (170). Cellular genes that produce mRNAs with upstream AUG codons often use alternative promoters and/or splice sites to generate supplementary transcripts in which the leader sequences are less problematical (68, 155,179,197,210,212,223,259,269). Indeed, the 5' variability is sometimes so extensive that no two cDNAs from a given gene have the same 5' noncoding sequence (145,155)! This underscores the need to distinguish between functional and nonfunctional (or minimally functional) cDNAs andmRNAs, a difficult problem that only a few investigators have tackled (3, 204). It might be mentioned in passing that, among growth control as well as housekeeping genes, G-Crich leader sequences are much more pervasive, and thus more ofa potential problem, than upstream AUG codons (23, 53, 77,89,134,136,138,163,166,178,191,229,231,252,270,280). In the case of the c-sis/PDGF-2 mRNA, which has both upstream AUG codons and a G-C-rich leader sequence, it is primarily the formation of secondary structure (implied 5. This number, which comes from reference 127, is probably inflated for reasons described in the next section of the text. 6. See reference 127 for additional documentation. by the G-C richness) that restricts translation in vivo (209). 7 This is noteworthy because the inhibitory effects of secondary structure may be susceptible to environmental modulation (129) while there is, as yet, no evidence that the inhibitory effects of upstream AUG codons are regulatable in vertebrates, as they are in the GCN4 gene of yeast (86, 220, 267a). In vertebrates, the solution to upstream AUG codons is to get rid of them (68, 155,179,197,210,212,223,259,269).

Lessons from cDNA Irregularities
Atypical or erroneous cDNA sequences have sometimes been mistaken as evidence against the generality of the scanning mechanism. Among cellular mRNAs that were initially reported to contain a slew of upstream AUG codons, many of the worst offenders have been exonerated as more data has emerged. For example, some long, AUG-burdened leader sequences have been recognized belatedly as errors in cloning or sequencing (58, 97, 183, 268; 52 corrected in 286; 59 corrected in 194; 110 corected in 153; 5 corrected in 56; 34 corrected in 103; 78 corrected in 139). 6 In some cases the error was simply that the cloned eDNA did not include the entire coding sequence: an internal AUG codon was mistakenly assumed to be the site of initiation, and therefore several upstream AUG codons, actually part of the coding sequence, were thought to burden the 5'-noncoding sequence (9 corrected in 79; 21 corrected in 22; 28 corrected in 94; 141 corrected in 142; 157 corrected in 254; 176 corrected in 175). The spurious upstream AUG codons in some cDNA sequences reflect derivation of the cDNA from a minor mRNA species that has an atypically long leader; the bulk of the transcripts from the same gene were shown to have a much shorter leader sequence, and no upstream AUG codons (95, 111, 151 corrected in 71). 6 As cloning efforts have gradually turned toward complicated regulatory genes, it has become fairly common to find cDNAs that correspond to incompletely processed transcripts (42, 63, 96,146,216,225,257). Thus, there are cases in which a bevy of out-of-frame AUG codons near the 5'-end of a cDNA sequence actually reside in an intron, which is not present, of course, in the functional mRNA (230, 236, 247, 272, 147 corrected in 64). For some Drosophila transcripts the excision of a 5'-intron, and consequent activation of translation, are developmentally regulated (17). If regulated (or merely inefficient) splicing is more widespread than we realize at present, most of the still-problematical eDNA sequences with multiple upstream AUG codons (e.g., 57, 83,108,177,186,206,256) might eventually be traced to intron-containing pre-mRNAs. There are some mammalian eDNA sequences with AUG-burdened leader sequences that have not yet been formally recognized as introns, but that possibility is consistent with their localization in the nucleus (20, 63, 170); or their inability to be translated unless the upstream AUG codons are removed (171,174,187); or the presence of a typical 3' splice junction motif at the point of divergence between two cDNA sequences (compare 154 with 260, 224 with 159, 135 with 152, and cDNAs a and b in reference 149); or the fact that an intron 7. In other cases (47, 107), the regulatory effect of leader sequences was demonstrated by deletion mutagenesis rather than site-directed mutagenesis; thus it is not clear whether an upstream AUG codon or the potential for secondary structure accounts for the observed inefficient translation of the wild-type sequence.
interrupts the 5'-noncoding sequence in other members of the gene family (compare 133 with 18, and 205 with 19). Many of the anomalous deductions about upstream AUG codons in mRNA sequences are favored, unfortunately, by the common practice of selecting and sequencing the longest cDNA clone! The main point here is not that cellular mRNA leader sequences never have upstream AUG codons but that such sequences are not nearly as common as superficial reading of the literature would suggest. When upstream AUG codons do occur they are a red flag, a clue to expect some sort of regulation (such as promoter switching or regulated splicing) or, at least, inefficient translation of the gene in question.

Artifacts of CeU-free Translation Systems
The results of some in vitro translation experiments have been taken as evidence against the generality of scanning. Five examples are often cited as evidence that eukaryotic ribosomes can bind directly to a site in the interior of a message without having traversed the upstream sequence. Rice et al. (213) have discussed the evidence for "internal initiation" in flaviviruses and have suggested credible alternative interpretations. As I cannot improve on their cogent discussion, I will simply recommend it to the reader and move on. For each of the other four examples, a synopsis of the arguments for and against internal initation follows.
(a) An SP6 vector-derived transcript corresponding to the adenovirus DNA polymerase gene directed the in vitro synthesis of the 120K (nearly full-sized) polymerase and a 62K polypeptide that mapped to the 3'-end of the P9] coding sequence (80). Because cDNA corresponding to the 5'-portion of ~ arrested the translation of the 120K but not the 62K product, the authors suggested that there is an independent ribosome-entry site at the midpoint of the ~ ORE and that the 62K protein was initiated internally. A less heretical explanation is that nuclease attack might have generated an mRNA fragment in which the initiator codon for 62K was near the 5'-end. The authors considered that explanation less likely because the translation results did not change when RNasin (an inhibitor of RNase) was included, and because 32p-labeled mRNA was only slightly degraded during the first 15 min of incubation in the reticulocyte extract. In retrospect, however, either RNA cleavage or some other artifact must have occurred in the in vitro experiments, because the 62K protein was not synthesized in vivo from a plasmid that produced an abundance of functional 140K DNA polymerase (237).
(b) When vesicular stomatitis virus mRNA P was translated in a reticulocyte extract, the products were the fullsized P protein and a 7K COOH-terminal fragment thereof (84). The 7K polypeptide was attributed to internal initiation based on hybrid arrest experiments; i.e., a cDNA fragment complementary to the Y-end of mRNA-P abolished translation of P but not the 7K protein. RNA cleavage was considered a less likely explanation because RNasin was present during the incubation and because the relative yield of 7K did not increase with time, as it might have were its template a degraded RNA. The second argument is weak, however, because one cleavage event might activate an mRNA fragment for 7K translation while the next clip, within the 7K coding sequence, might inactivate it. RNasin is not adequate insurance, as proven by the adenovirus story. The fact that cap analogues did not inhibit 7K translation (85) is equally consistent with internal initiation or initiation at the 5'-end of a broken template. A strong hint that the 7K protein is an artifact came from an experiment in which a transcript representing only the Y-portion of the P gene (derived by subcloning that region into an SP6 vector) was translated in vitro: three small polypeptides were made, and the two "nonphysiological" products were more abundant than the "authentic 7K band (84). Virus-infected cells contain barely detectable amounts of a protein that does not exactly comigrate with the 7K in vitro band (84) and might itself be a degradation product.
(c) In the case of infectious pancreatic necrosis virus, the claim (162) is that genomic RNA-A directs the independent synthesis of three major proteins: VP2, NS, and VP3. There is no absolute need to postulate three initiation events inasmuch as the sequence of genome segment A reveals a single ORF that encodes VP2-NS-VP3 as a fusion protein (49), and a protease activity that maps with NS is able to release the mature proteins from the common precursor (50). Thus the speculation is that, in addition to the polyprotein mode of translation, NS and VP3 can be translated by a second mechanism which involves internal initiation. The main supporting evidence seems to be that, during a short pulse with [35S]methionine in vitro, VP2, NS, and VP3 acquired label simultaneously (162). But that observation is equally consistent with independent internal initiation of three proteins from one mRNA or translation of each protein from an independent template, generated by RNA degradation. In addition to VP2, NS, and VP3, in vitro translation of RNA-A produced countless other polypeptides that are never seen in vivo (50, 162), which is strong reason to suspect an in vitro artifact. The NH2-terminal amino acid sequence of virionderived VP3 has not yet been analyzed to ascertain the presence of either methionine, which would be consistent with internal initiation, or a protease-recognition sequence, which would implicate a polyprotein as the sole source of VP3 in vivo. Lacking such evidence, the argument for internal initiation is weak.
(d) The last example is poliovirus. When RNA from virions was translated in a reticulocyte lysate, the earliest detected products mapped to the 3' portion of the genome (region P3), suggesting internal initiation site(s) (200). In support of that idea, N-formyl[35S]methionine (a marker of initiation) was incorporated into polypeptides from region P3 (46). The question is whether internal start sites are accessible to ribosomes in intact poliovirus mRNA or whether RNA fragments are the functional templates. The anomalous internal sites detected in the reticulocyte system did not function in HeLa cell extracts, nor when an aliquot of HeLa cell extract was added to the reticulocyte lysate. The authors' interpretation was that HeLa cell extracts contain undefined factors that promote 5' initiation, and "the deficiency (of those factors in the reticulocyte system) resulted in the ability of ribosomes to initiate translation on internal sequences" (46). One might think that deficiency of a required component would preclude translation, rather than endow ribosomes with a novel power; but that is debatable. The fact is that no viruspromoting initiation factor has yet been purified from HeLa cells, but HeLa extracts have been shown to contain an RNase inhibitor (249)! Finally, an experiment that might have detected internal initiation in vivo failed to do so. The experi-ment involved poliovirus mutants in which small changes in the 5' non-coding sequence depressed translation from the normal 5' proximal site (265). Were there an independent initiation site in the interior of the poliovirus genome, it should have remained functional; but the residual level of translation in mutant-infected cells showed no enrichment for P3 products. Thus, although the mechanism of initiation at the 5' proximal site in poliovirus mRNA remains unclear, the existence of an internal initiation site in the 3' third of the genome can almost certainly be dismissed as an artifact.
Curran and Kolakofsky (38) have interpreted some recent results from the Sendai virus system as evidence that ribosomes bind to the 5' end of the "P" transcript and jump some 1,500 nucleotides to the start of the "X" ORE thus translating the X protein by a cap-dependent, scanning-independent process. Their hypothesis is based largely on the ability of cap analogues to inhibit X synthesis, but the hypothesis is contradicted by the virtual absence of X translation in vitro unless the P transcript is cleaved! In view of the many precedents for activation of internal initiation sites by mRNA cleavage (13, ll6, 140, 195)and for inhibition of translation of uncapped transcripts by cap analogues (15, 61, 100,227,246), the interpretation offered by Curran and Kolakofsky seems unwarranted.
The main point here is not to debunk a few spurious claims against the generality of scanning but to illustrate the need for caution whenever cell-free systems are used to study translation. The tendency to see "internal initiation" more often in reticulocyte (12,14,40,73,180,238,264,271,279,284) than in wheat germ translation systems might simply reflect the greater ease of translating broken (hence uncapped) mRNAs in the former system. A final caution is that initiation at non-AUG codons occurs far more efficiently in vitro than in vivo (4, 74,192,275) and therefore cell-free systems are not a reliable way to explore the rare, interesting situations in which eukaryotic ribosomes seem to initiate at codons other than AUG (10, 11, 37, 74). The usual failure of eukaryotic ribosomes to initiate at non-AUG codons in vivo is illustrated by the ability of point mutations in the AUG initiator codon to abolish gene expression (27,32,44,72,182,203,211,217,258). Conversely, bacterial genes that initiate with a GUG codon require the substitution of an AUG codon for successful expression in mammalian cells 037, 240).

Putting the Steps Together
Here is a brief statement of what we know and what we have yet to learn about the first three steps in initiation: binding of the 40S-ribosome/Met-tRNA/factor complex to mRNA; scanning; and recognition of the AUG initiator codon.
Step 1 The ubiquitous m7G cap and the associated cap-binding protein(s) (233) explain the predilection of eukaryotic ribosomes to engage mRNAs at the 5'-end. The initiation mechanism is end dependent even in those uncommon instances in which a cap is absent, however, prompting the idea that the 40S subunit might thread onto the 5'-end of mRNA (113). Microscopic data obtained with a new image processing technique indeed suggests the possibility of a channel running through the neck region of the 40S ribosomal subunit (60) which could be the needle's eye. (If the scanning mecha-nism were really a threading mechanism, the ability of 40S ribosomal subunits to hold on and reinitiate downstream might almost have been expected!) Apart from cap-recognition factor(s), we do not understand the precise function of any of the initiation factors that mediate early step(s) in the binding of mRNA to ribosomes. The remarkable finding (45) that a point mutation in eIF2 (the Met-tRNA~met-binding factor) affects where ribosomes initiate on mRNA illustrates the potential for surprises in this area.

Step2
All we yet know about the mechanism of 40S subunit locomotion is that ATP is required (115), in common with other proteins that bind to nucleic acids and then (in an equally unknown manner) slide (25, 54). Scanning seems to be facilitated by something in the cytoplasm of mammalian cells, either a soluble protein or a 40S ribosome-associated activity, that has considerable ability to unwind duplex structures in the 5'-noncoding region of mRNA (123). The 40S subunit apparently migrates linearly, as evidenced by the ability of an upstream AUG codon to completely suppress initiation from a downstream site, even when the downstream AUG codon occurs in the same context as, and only five nucleotides beyond, the first AUG triplet (Fig. 7 in reference 121). The inability of ribosomes to "jump" a hairpin structure that is too stable to be melted is further evidence of the linearity of scanning (123). We do not know whether the 40S ribosome advances nucleotide-by-nucleotide or triplet-by-triplet; but, if it is the latter, each entering ribosome must pick at random any one of the three possible frames. If scanning were uniquely phased, a one-or two-nucleotide insertion between the cap and the AUG codon should drastically impair translation; in fact, such mutations are usually innocuous. An interesting possibility is that 40S ribosomal subunits might be nudged into the correct phase by the GCC or ACC motif that immediately precedes the AUG codon in vertebrate mRNAs. That would explain the ability of ~CC to enhance initiation when the purine occurs in position -3 or (less effectively) position -6, but not when the ~CC motif is shifted out-of-phase with respect to the AUG codon (122,126). The observation that increasing the length of the leader sequence never impairs (and sometimes enhances) translation (129) suggests that, at least in the absence of secondary structure, scanning is not the rate limiting step in initiation.

Step3
The migrating 40S ribosomal subunit stalls at the first AUG codon, which is recognized in large part by base pairing with the anticodon in Met-tRNA~ ~"°t (30). The stop-seanning step, and hence selection of the initiator codon, is susceptible to modulation, however: by context, at least in vertebrates (122,126); by mutant forms of yeast eIF2 that compensate for (stabilize?) a weakened codon/anticodon interaction (45); by the antibiotic edeine (131); and by varying the concentration of Mg 2+ and other ions in cell-free translation systems (114,192,221,275).

Implications, Limitations, and Alternatives
In addition to correctly predicting the start sites for translation in the majority of viral and cellular mRNAs, the scanning model has informed the design of a variety of experi-ments, including the construction of mammalian vectors that can initiate translation in all three reading frames (169) and the development of a "translational assay" for monitoring immunoglobulin gene rearrangements (55). The scanning mechanism explains the considerable effort expended by viruses (125) and some cells (93) to convert polycistronic transcripts into translatable (monocistronic) mRNAs; rationalizes an alternative strategy where several (up to seven!) cistrons are fused to generate some remarkable multifunctional proteins (62, 82,98,158); and justifies the observed inefficient translation of some critical genes, including some protooncogenes (156,204,209). The inhibitory effect of upstream AUG codons has been incorporated into an interesting model regarding the mechanism of allelic exclusion (143).
The scanning model is compatible with various modes of negative regulation, such as inhibition by upstream AUG codons (86, 267a), or by a repressor protein (219), or by secondary structure within the 5'-noncoding region; but the scanning mechanism seems incompatible with intricate schemes for enhancing the translation of specific mRNAs. Indeed, apart from the ubiquitous m7G cap and the conserved sequence around the initiator codon, the only other defined feature that has been shown to increase translational efficiency in eukaryotes is a long, unstructured 5'-sequence (129). Although leader-shuffling experiments have documented the ability of certain cellular and viral 5'-sequences to support efficient translation (16, 67, 106,150,253,278), the application of mutagenesis techniques has consistently failed to pinpoint an effector motif within such leader sequences (160,242). Thus, the data so far support the prediction (125) that, beyond the m7G cap and a favorable context near the AUG codon, what makes a "good" 5'-noncoding sequence is merely the absence of any unfavorable features.
An alternative to the three-step initiation mechanism described above is that 40S ribosomal subunits might bind directly to an internal site in some rare mRNAs. Although no credible evidence yet supports that possibility, it can never be ruled out. Neither will it ever become a fact unless the deficiencies in current experiments are recognized. One cannot ignore mRNA degradation in vitro as if it were a minor irritation. In vivo experiments have other limitations. The most carefully executed Northern assays sometimes fail to detect transcripts which, genetic evidence tells us, must be present and functional in cells (10). When it comes to reporter genes, the more sensitive the assay for protein expression, the harder it is to pinpoint the functional mRNA. Thus, a recent report of supposedly efficient expression of adenosine deaminase from the 3'-end of a dicistronic vector (161) was interpreted more cautiously when the high specific activity of adenosine deaminase was taken into account (184).
The existence of credible rules for initiation calls attention to occasional mRNAs that break the rules. In fact, very few viral mRNAs remain incompletely understood. It seemed for a while as ifpoxviruses might follow different rules for translation inasmuch as, in nearly all of the viral late genes, the ATG initiator codon is preceded at the DNA level by a T in position -3 (218, 276); we now know, however, that a complicated transcriptional mechanism replaces the undesirable T with the preferred A in position -3 (190,226). I still cannot explain the translation of picornaviruses, cauliflower mosaic virus (43), or the putative bifunctional mRNAs from Epstein Barr virus (274) and cottontail rabbit papillomavirus (7). In the last two cases, however, the transcription patterns are complex and it seems possible that the downstream protein is translated, not from the 3'-end of the recognized bicistronic mRNA, but from a scarce monocistronic mRNA that has not yet been detected. One way to rationalize the peculiar leader sequences on retrovirus and poliovirus mRNAs is to suppose that feature~ that compromise translation are tolerated because those very features are required to replicate (33) and package the viral genome (1). Moreover, viruses can and do compensate for inefficient translation by using their efficient transcription signals to flood the cell with mRNA (125). As for the structure of cellular genes, I have argued herein that the presence of numerous upstream AUG codons in a cDNA sequence constitutes a strong hint that the cDNA might represent an intron-containing pre-mRNA, rather than the funtional mRNA from the gene in question. The growing number of such sequences in the vertebrate cDNA catalogue raises the interesting possibility that the final, regulated step in the expression of many critical genes is the conversion of a stable, untranslatable precursor to a functional mRNA. Identification of a family of muscarinic acetyleholine receptor genes.