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Colloquium

Molecular mechanisms of translation initiation in eukaryotes
Tatyana V. Pestova*, Victoria G. Kolupaeva*, Ivan B. Lomakin*, Evgeny V. Pilipenko*§, Ivan N. Shatsky, Vadim I. Agol, and Christopher U. T. Hellen*¶
*Department of Microbiology and Immunology, State University of New York Health Science Center at Brooklyn, Brooklyn, NY 11203; A. N. Belozersky Institute of Physico-chemical Biology, Moscow State University, Moscow 119899, Russia; and Institute of Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences, Moscow Region 142782, Russia

Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2 GTP Met-tRNAi and eIF3) to the 5 end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A 4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.

(iv) Displacement of factors f rom the 48S c omplex and join ing of the 60S subun it to for m an 80S ribosome, leav ing Met-tRNAi in the ribosomal P site. Research in our laborator y has addressed the molecular mechan isms of these dif ferent st ages in translation in itiation and the means by which they are bypassed during in itiation by internal ribosomal entr y. We have rec onstituted each of these st ages in vitro using purified translation c omponents to identif y the min imum set of eIFs that is required for each st age and to prov ide a f ramework for more det ailed mechan istic analysis.
Factor Requirements for Ribosomal Attachment and Scanning of 43S Ribosomal Complexes on -Globin mRNA. The in itiation c odon of a

ranslation of mRNA into protein begins af ter assembly of in itiator tRNA (Met-tRNAi), mRNA, and separated 40S and 60S ribosomal subun its into an 80S ribosome in which MettRNAi is positioned in the ribosomal P site at the in itiation c odon. The c omplex in itiation process that leads to 80S ribosome for mation c onsists of several linked st ages that are mediated by euk ar yotic in itiation factors. These st ages are: (i) Selection of in itiator tRNA f rom the pool of elongator tRNAs by euk ar yotic in itiation factor (eIF)2 and binding of an eIF2 GT P Met-tRNAi ternar y c omplex and other eIFs to the 40S subun it to for m a 43S prein itiation c omplex. (ii) Binding of the 43S c omplex to mRNA, which in most inst ances oc curs by a mechan ism that involves in itial rec ogn ition of the m7G cap at the mRNA 5 -ter minus by the eIF4E (cap-binding) subun it of eIF4F. Ribosomes bind to a subset of cellular and v iral mRNAs as a result of cap- and endindependent internal ribosomal entr y. (iii) Movement of the mRNA-bound ribosomal c omplex along the 5 nontranslated region (5 NTR) f rom its in itial binding site to the in itiation c odon to for m a 48S in itiation c omplex in which the in itiation c odon is base paired to the antic odon of in itiator tRNA.
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This paper was presented at the National Academy of Sciences colloquium, ``Molecular Kinesis in Cellular Function and Plasticity,'' held December 79, 2000, at the Arnold and Mabel Beckman Center in Irvine, CA. Abbreviations: eIF, eukaryotic initiation factor; 5 NTR, 5 nontranslated region; IRES, internal ribosomal entry site; EMCV, encephalomyocarditis virus; FMDV, foot-and-mouth disease virus; TMEV, Theiler's murine encephalitis virus; BVDV, bovine viral diarrhea virus; CSFV, classical swine fever virus; HCV, hepatitis C virus; ITAF, IRES transactivating factors; PTB, pyrimidine tract-binding protein; RRL, rabbit reticulocyte lysate; GMP-PNP, guanosine 5 -[ , -imido]triphosphate.
§

Present address: Department of Neurology, University of Chicago Medical Center, Chicago, IL 60637. To whom reprint requests should be addressed at: Department of Microbiology and Immunology, State University of New York Health Science Center at Brooklyn, 450 Clarkson Avenue, Box 44, Brooklyn, NY 11203. E-mail: chellen@netmail.hscbkyn.edu.

¶

PNAS

June 19, 2001

vol. 98

no. 13

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euk ar yotic mRNA is nor mally the first AUG triplet downstream of the 5 -ter minal cap and is usually separated f rom it by 50 100 nt. Af ter cap-mediated att achment to mRNA, a 43S c omplex is thought to scan downstream f rom the 5 -end until it enc ounters the in itiation c odon. We used native capped -globin mRNA as a model in in vitro rec onstitution ex periments to address three basic questions. (i) Which eIFs are required for a 43S c omplex to bind capped mRNA? (ii) Which eIFs are required for the bound c omplex to move downstream to the in itiation c odon? (iii) How does the scann ing 43S c omplex rec ogn ize and reject mismatched interactions bet ween the Met-tRNAi antic odon, and triplets in the 5 NTR until the c orrect in itiation c odon is reached and rec ogn ized? In these ex periments, the position of the leading edge of bound ribosomal c omplexes on mRNA was mapped by primer extension inhibition (``toeprinting''). The estimated length of the mRNA-binding clef t in 40S subun its is 30nt, and 48S c omplexes usually yield toeprints at positions 15 17 downstream of the A of the in itiation c odon. Ribosomal binding at the 5 -end of the mRNA required eIF3, the eIF2 GT P Met-tRNAi c omplex, AT P, and the eIF4F capbinding c omplex, and was enhanced by eIF4B (1). eIF4F is a heterotrimeric factor, and its eIF4A (AT P-dependent RNA helicase) and eIF4E subun its and the eIF4G550 1090 f ragment of its 1,560-amino acid eIF4G subun it c onstitute the c ore of eIF4F

Fig. 1. The mechanism of action of eIF1 and eIF1A in promoting assembly of 48S ribosomal complexes at the authentic initiation codon of a conventional capped mRNA. The 5 terminal m7G residue is shown as a filled black circle, the 5 NTR as a black line, and the ORF downstream of the AUG initiation codon as a black rectangle. (A) In the presence of eIFs 2, 3, 4A, 4B, and 4F, an aberrant ribosomal complex (``complex I'') assembles at a cap-proximal position but is unable to scan downstream to the initiation codon. (B) In the presence of eIFs 1, 1A, 2, 3, 4A, 4B, and 4F, 48S ribosomal complexes assemble exclusively at the authentic initiation codon. (C) Addition of eIF1 and eIF1A to complex I promotes its complete conversion to correctly assembled 48S complexes after dissociation of complex I and rebinding of 43S ribosomal complexes in a scanning-competent form.

suf ficient for ef ficient ribosomal att achment to capped mRNA (2). This f ragment of eIF4G binds both eIF4E and eIF4A and probably c oordinates their activ ities so that a cap-prox imal region of mRNA is unwound and is thus rendered ac cessible to an inc oming 43S c omplex so it can bind productively. The molecular interactions that enable the inc oming 43S c omplex to bind this ``prepared'' template are not k nown but are thought to involve interaction of the eIF3 c omponent of 43S c omplexes w ith cap-associated eIF4G. The bound ribosomal ``c omplex I'' was arrested in a cap-prox imal position and did not reach the in itiation c odon (Fig. 1A). Two additional activ ities present in rabbit reticuloc y te lysate (RR L) enabled 43S c omplexes to reach the in itiation c odon, for ming ``c omplex II'' w ithout being arrested at the in itial binding site (Fig. 1B). These small factors were purified and identified by sequencing as eIF1 (13.5 kDa) and eIF1A (19 kDa) and c ould be functionally replaced by c orresponding rec ombinant polypeptides. These t wo factors acted synergistically; eIF1A w ithout eIF1 enhanced eIF4F-mediated binding of 43S c omplexes to mRNA but did not enable these c omplexes to reach the in itiation c odon, whereas eIF1 w ithout eIF1A reduced the prominence of the cap-prox imal c omplex I and promoted formation of low levels of 48S c omplexes. The interaction w ith mRNA of 48S c omplexes assembled in the absence of eIF1A dif fered subtly f rom c omplexes for med in their presence, in that only t wo ( 16 17) rather than three toeprints ( 15 17) were apparent. eIF1A therefore increases the c ompetence of 43S c omplexes to bind mRNA and the processiv it y of scann ing
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43S eIF1 mRNA c omplexes. eIF1A also st abilizes binding of the ternar y c omplex to 40S subun its in the absence of mRNA (3, 4), presumably by an allosteric mechan ism, because it is not k nown to interact directly w ith any c omponent of the ternar y c omplex. This st abilization by eIF1A is weak but might be indicative of a role for eIF1A in ensuring that in itiator tRNA and mRNA adopt the c orrect relative orient ation on the scann ing ribosomal c omplex. eIF1A c omprises an oligonucleotide-binding (OB) -barrel fold that closely resembles prok ar yotic in itiation factor IF1 (and c orresponds to the region of sequence homolog y bet ween them) and an additional C-ter minal domain (4). The ex periment ally deter mined RNA-binding sur face of eIF1A is large, extending over the OB fold and the adjacent groove leading to the sec ond domain. Mut ations at multiple positions on this sur face resulted in a reduced abilit y of eIF1A to promote assembly of 48S in itiation c omplexes at the in itiation c odon. The RNA ligand for eIF1A is not k nown, but by analog y w ith IF1 (5), eIF1A might bind 18S ribosomal RNA in the ribosomal A site. In the absence of eIF1 and eIF1A, the mRNA-binding clef t on 40S subun its appears to be open, because they can bind mRNA in an end-independent manner during in itiation by internal ribosomal entr y (see below). eIF1 and eIF1A may c ontribute to the c orrect interactions of c omponents of the 43S c omplex w ith mRNA that enable it to enter a processive mode, for example by closing this clef t directly or indirectly and possibly even by for ming part of the channel on the 40S subun it through which mRNA moves during ribosomal scann ing. Ex periments done by using c ompetitor mRNAs indicated that c omplex I cannot be ``chased'' directly into c omplex II and is therefore not its immediate precursor. Complex I is aberrantly assembled (because it is arrested at a non-AUG triplet and is unable to scan to the in itiation c odon) and is intrinsically unst able. eIF1 and eIF1A together (but not indiv idually) promote dissociation of c omplex I and enable the released 43S c omplex to rebind mRNA in a c ompetent st ate to scan to the in itiation c odon (Fig. 1C). eIF1 alone is able to rec ogn ize and dest abilize ribosomal c omplexes inc orrectly assembled by internal ribosomal entr y (see below). Identification of this activ it y of mammalian eIF1 is c onsistent w ith characterization of its yeast homologue Sui1 as a mon itor of translation ac curac y. Mut ations in Sui1 allow aberrant in itiation in vivo at non-AUG c odons by mismatch base pairing w ith Met-tRNAi (e.g., ref. 6). Deter mination of the solution str ucture of eIF1 by NMR (7) has revealed that these mut ated residues for m part of a sur face that is almost per fectly c onser ved among all eIF1 homologues and that is likely directly involved in in itiation c odon selection by eIF1. In summar y, we have deter mined the set of factors required for binding of a 43S c omplex to a model native capped mRNA and for it to scan to the in itiation c odon. These ex periments were done by using -globin mRNA, and it is possible that ribosomal scann ing on longer or more highly str uctured 5 NTRs may require additional as-yet-un identified factors, for example to enhance processiv it y or to promote unw inding of st able sec ondar y str uctures. A lmost all aspects of the mechan ism of ribosomal scann ing remain uncharacterized (8). For example, scann ing is an AT P-dependent process, but it is not k nown whether ribosomal movement itself involves hydrolysis of AT P or whether chemical energ y is required only to unw ind sec ondar y str ucture in the 5 NTR to per mit ribosomal movement by onedimensional dif fusion f rom its in itial 5 -ter minal att achment site. The abilit y to rec onstitute this process in vitro w ill enable this and other outst anding questions to be addressed.
Factor Displacement from the 48S Complex and Joining to a 60S Subunit to Form Active 80S Ribosomes. The 48S c omplex assembled

at the in itiation c odon of -globin mRNA is bound by factors that must be displaced before the 40S subun it mRNA MetPestova et al.

tRNAi c omplex can join w ith a 60S subun it. Substitution of GT P by guanosine 5 -[ , -imido]triphosphate (GMP-PNP) (a nonhydrolyzable analogue) arrests in itiation at the st age of 48S c omplex for mation, indicating that displacement of factors and subun it join ing both require hydrolysis of GT P bound to eIF2 in 48S c omplexes. GT P hydrolysis by eIF2 is activated by eIF5, a 49-kDa polypeptide that interacts specifically w ith eIF2 and eIF3 (9, 10). Recent dat a suggest that eIF5 is a c omponent of multifactor c omplex c omprising eIF1, eIF3, eIF5, and the eIF2 GT P MettRNAi ternar y c omplex that can ex ist f ree of the ribosome and probably binds to it as a whole rather than sequentially (10). eIF5 binds strongly to eIF2 but induces its GT Pase activ it y only when eIF2 is associated w ith the 40S subun it. GT P hydrolysis, which leads to dissociation of eIF2-GDP, is thought to be induced in response to base pairing bet ween the in itiation c odon and the antic odon of Met-tRNAi, thereby ensuring stringent selection of the in itiation c odon during the scann ing process (11). Until recently, the hydrolysis of eIF2-bound GT P was c onsidered the only requirement for the join ing of a 60S subun it to the 48S c omplex (see ref. 12 for a rev iew). However, we found that addition of 60S subun its and rec ombinant eIF5 to 48S c omplexes assembled on globin mRNA did not lead to for mation of 80S ribosomes (13). A partially purified ribosomal salt wash f raction f rom mouse ascites cells was active in promoting 80S ribosome assembly and was therefore used as a source for purification of additional factors. We purified t wo proteins to apparent homogeneit y, which together but not separately were able to mediate assembly of 48S c omplexes and 60S subun its into 80S ribosomes. The smaller (49 kDa) protein c ould be functionally replaced by rec ombinant eIF5. The sec ond protein had an apparent molecular mass of 175 kDa, and its N-ter minal sequence identified it as a mouse homologue of prok ar yotic in itiation factor IF2 (Fig. 2). A role for a euk ar yotic homologue of IF2 was first revealed by studies in yeast (14). A nalysis of poly ribosome profiles showed that deletion of the yeast IF2 homologue led to a reduction in for mation of larger polysomes and an ac cumulation of inactive 80S ribosomal particles, and in vitro translation assays c onfir med that this deletion led to a defect in translation in itiation on the majorit y, if not all, cellular mRNAs. This defect c ould be rescued by adding back purified rec ombinant protein (14). These results indicated that this protein is a general translation factor in yeast. Human, Drosophila, and archaeal homologues have also been identified (15, 16). In light of its function in subun it join ing, we named this factor eIF5B (13). Rec ombinant human eIF5B5871220 lack ing amino acids 1586 c ould substitute for yeast eIF5B in vivo (15) and for native mammalian eIF5B in subun it join ing in our in vitro rec onstitution ex periments (13). It is almost cert ain that eIF5B is a protein that was prev iously implicated in subun it join ing but subsequently erroneously disc ounted as an inactive c ont aminant of eIF5 (12). Puromycin resembles the 3 -end of aminoac ylated tRNA and can bind to the ribosomal A site to react w ith Met-tRNAi in the P site to for m methionylpuromycin. This reaction mimics formation of the first peptide bond, and we therefore used it to c onfir m that 80S ribosomes assembled by using eIF5 and eIF5B were active. Assembly of 48S c omplexes on AUG triplets is much simpler than on native mRNA, because it involves neither 5 -end-dependent att achment nor scann ing. 48S c omplex formation on AUG triplets requires only a 40S subun it and the eIF2 GT P Met-tRNAi c omplex, which enabled us to investigate the inf luence of other factors on the requirements for subun it join ing (13). A requirement for both eIF5 and eIF5B for 80S assembly was apparent only when 48S c omplexes were assembled by using eIF1, eIF1A, eIF2, and eIF3. These four factors are all nor mally associated w ith a 48S c omplex at the in itiation c odon. Indiv idually, eIF5 and eIF5B were equally active in subun it
Pestova et al.

Fig. 2. Sequence and structural conservation of eukaryotic eIF5B proteins from Homo sapiens (15), Drosophila melanogaster (16), and Saccharomyces cerevisiae (14), archaeal IF2 from Methanococcus jannaschii and prokaryotic IF2 from Escherichia coli (12). The percentages of amino acid identities to human eIF5B in the N-terminal region of the protein, the GTP-binding domain, and the C-terminal region of the protein are shown. The black rectangle in the schematic representation identifies the position of the GTP-binding domains in these proteins with the indicated GTP-binding protein consensus sequence motifs G1-G5 aligned with sequence motifs G1-G5 of E. coli IF2 and human eIF5B. Numbers above the domains of eIF5B IF2 proteins refer to the amino acid residues in each protein; numbers below the aligned sequences refer to the amino acid residues in G1G5 motifs of human eIF5B.

join ing in reactions lack ing eIF1 and eIF3, but inclusion of eIF1 and eIF3 together reduced the indiv idual activ ities of both eIF5 and eIF5B. The requirement for both eIF5 and eIF5B in these circumst ances indicates that they have c omplement ar y functions. Hydrolysis of GT P bound to 48S c omplexes is a prerequisite for subun it join ing and was therefore also c ompared in the presence and absence of eIF1 and eIF3 (13). eIF5 and eIF5B stimulated GT P hydrolysis by eIF2 equally when 48S c omplexes c ont ained only eIF1A and eIF2, but inclusion of eIF1 and eIF3 inhibited the stimulator y activ it y of eIF5B w ithout af fecting that of eIF5. This ef fect can ac c ount for the reduced abilit y of eIF5B to promote methionylpuromycin synthesis in the presence of eIF1 and eIF3. We c onclude that, although eIF5 is active in inducing GT P hydrolysis on 48S c omplexes in the presence of a full set of factors (including eIF1 and eIF3), this is insuf ficient for subun it join ing. Under these circumst ances (when all factors associated w ith 48S c omplexes are present, which c orresponds to the nor mal situation for in itiation on capped mRNAs), eIF5B is also required. The central domain of eIF5B c ont ains sequence motifs characteristic of GT P-binding proteins (Fig. 2). By U V crosslink ing, we found that [32P]GT P bound directly to eIF5B independently of ribosomal subun its, and that bound [32P]GT P exchanged readily w ith unlabeled GT P, GMP-PNP, or GDP. eIF5B had no detect able intrinsic GT Pase activ it y, but its abilit y to hydrolyze GT P was activated by 60S subun its and c onsiderably more by 40S and 60S subun its together. Interestingly, prok ar yotic IF2 is also
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a GT Pase that is specifically activated by large and small ribosomal subun its together (17). This similarit y bet ween the homologous factors eIF5B and IF2 suggests that ribosomal activation of their GT Pase activ it y may oc cur by a c ommon mechan ism. Binding of GT P to eIF5B may be required for it to adopt an active c onfor mation. To test this hypothesis, 48S c omplexes were assembled w ith GT P, separated f rom un inc orporated GT P by gel filtration, and then incubated w ith eIF5, 60S subun its, dif ferent nucleotides, and either full-length native eIF5B or rec ombinant eIF5B5871220. The deg ree of dependence of eIF5B's activ it y in 80S assembly on binding GT P was deter mined by the integrit y of the protein. eIF5B5871220 was c ompletely GT P-dependent, whereas native eIF5B ret ained low activ it y in the absence of GT P but was nevertheless stimulated 3-fold by GT P (T.V.P., unpublished work). This result suggests that eIF5B adopts the active c onfor mation required for subun it join ing when it binds GT P. eIF5B acts cat aly tically in the presence of GT P, promoting multiple rounds of subun it join ing. 80S c omplexe s were also for med by eIF5B bound to GMPPNP, but eIF5B-GMP-PNP acted stoichiometrically rather than cat aly tically. This defect in the activ it y of eIF5B in the presence of GMP-PNP c ould be because hydrolysis of GT P bound to eIF5B is required for the release of eIF5B f rom assembled 80S ribosomes, for the release of other factors, or for both. The proportion of Met-tRNAi in 80S ribosomes assembled in the presence of GT P (60%) that reacted w ith puromycin was sign ificantly greater than in c omplexes assembled by using GMP-PNP (8%). Methionylpuromycin synthesis by purified 80S ribosomes assembled in the presence of GT P was c ompletely inhibited by addition of eIF5B5871220 w ith GMP-PNP but not by either eIF5B5871220 or GMP-PNP alone. This result indicates that eIF5B-GMP-PNP can interact w ith preassembled 80S c omplexes, block ing their abilit y to react w ith puromycin (13). The specific inhibition of this reaction suggests that eIF5B binds to the ribosomal A site. When ribosomal c omplexes assembled by using GT P were resolved on sucrose densit y gradients, no eIF5B5871220 was detected on 40S, 48S, 60S, or 80S c omplexes. However, a large amount of eIF5B5871220 was bound to 80S c omplexes assembled in the presence of GMP-PNP. The inabilit y of eIF5B5871220 to hydrolyze GMP-PNP therefore locks the factor on 80S c omplexes and renders them inactive in methionylpuromycin synthesis. eIF1, eIF2, and eIF3 were detected in 48S c omplexes but not in 80S c omplexes assembled w ith GT P or GMP-PNP. GT P hydrolysis by eIF5B is therefore not required for the release of these factors during subun it join ing but is needed for release of eIF5B itself. The inabilit y of eIF5B5871220 GMP-PNP to dissociate f rom 80S ribosomes ex plains the requirement for stoichiometric rather than cat aly tic amounts of this factor in assembly reactions in the presence of GMP-PNP. Neither the st age during the in itiation process at which eIF1, eIF1A, eIF2, eIF3, and eIF5 are released nor the mechan ism by which release oc curs during in itiation on native mRNAs has yet been est ablished. Ribosomal subun it join ing to for m active 80S ribosomes that are c ompetent to begin elongation therefore involves t wo successive GT P hydrolysis events: activation by eIF5 of hydrolysis of eIF2-bound GT P and ribosome-activated hydrolysis of eIF5Bbound GT P. Remark ably, eIF5B is a homologue of prok ar yotic IF2, which also mediates a similar subun it-join ing step that also involves ribosome-activated hydrolysis of factor-bound GT P.
Initiation of Picornavirus Translation by Internal Ribosomal Entry: The Role of Canonical Initiation Factors. Pic ornav ir us RNA genomes

are uncapped and have highly str uctured 5 barriers to scann ing ribosomes. In itiation on end-independent and is instead mediated by a ribosomal entr y site (IR ES) in the 5 NTR (18,
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NTRs that are these mRNAs is 400-nt internal 19). The activ it y

of an IR ES depends on its str uctural integrit y, and even point mut ations can cause general or cell t ype-specific loss of function. Pic ornav ir us IR ESs are div ided into t wo major groups on the basis of sequence and str uctural similarities (20, 21). One group c ont ains poliov ir us and rhinov ir us, and the other group c ont ains encephalomyocarditis v ir us (EMCV), Theiler's murine encephalomyelitis v ir us (TMEV), and foot-and-mouth disease v ir us (FMDV). The EMCV and TMEV in itiation c odons are located at the 3 border of the IR ES, and ribosomes bind directly to them w ithout scann ing (22, 23). In poliov ir us, the in itiation c odon is 160 nt f rom the 3 border of the IR ES, and it is possible that the ribosome reaches it either by scann ing or by disc ontinuous transfer (``shunting'') af ter in itial att achment to the IR ES (24). Pic ornav ir us infection of ten leads to shutof f of cap-mediated translation in itiation, for example by rhinov ir us protease cleavage of eIF4G at R641 G642, such that the N-ter minal domain of eIF4G that binds eIF4E and the poly(A)-binding protein is separated f rom the C-ter minal domain that binds eIF3 and eIF4A (25). This cleavage impairs eIF4F's function in in itiation on capped mRNAs. However, as described below, this cleavage yields a f ragment of eIF4F that ret ains functions necessar y for pic ornav ir us IR ES-mediated in itiation. We rec onstituted in itiation in vitro on the EMCV IR ES and found that it is AT P-dependent and requires only eIFs 2, 3, and either eIF4F or eIF4A and the central third of eIF4G to which eIF4A binds (26, 27). The requirement for eIF4A and the c ognate domain of eIF4G is c onsistent w ith the profound inhibition of EMCV translation caused by dominant negative eIF4A mut ant polypeptides (28). The inhibition caused by these mut ants is thought to be because of their failure to ex it the eIF4F c omplex and rec ycle ef ficiently, thereby trapping it in an inactive for m. In this model, eIF4A therefore plays its role in in itiation as part of a c omplex w ith eIF4G rather than as a singular polypeptide. 48S c omplex for mation was enhanced 4-fold by eIF4B and less than 2-fold by the py rimidine tract-binding protein (P TB), a noncanon ical mRNA-specific in itiation factor (see below). Together, these factors promoted 48S c omplex for mation equally at AUG834 (the authentic in itiation c odon) and at AUG826 (which is v irtually unused in vivo). Remark ably, inclusion of eIF1 in assembly reactions or even addition of eIF1 to prefor med c omplexes led to dissociation of the ribosomal c omplex at AUG826 (13). This obser vation is c onsistent w ith the prev iously noted function of eIF1 in enhancing the fidelit y of in itiation c odon selection. The principal dif ference bet ween the factor requirements for in itiation on -globin mRNA and on the EMCV IR ES is that the latter has no requirement for eIF4E or the f ragment of eIF4G to which it binds and is therefore not impaired by cleavage of eIF4G by v iral proteases. eIF4E is a major focus of mechan isms that regulate in itiation of translation in vivo (29). The EMCV IR ES and other IR ESs that do not require eIF4E are therefore active in circumst ances that lead to inhibition of cap-mediated in itiation by impair ment of eIF4E function. A sign ificant insight to the mechan ism of in itiation on the EMCV IR ES came f rom the obser vations that eIF4F bound to the J-K domain of the EMCV IR ES a little upstream of the in itiation c odon (Fig. 3), and that this interaction is essential for in itiation (26). The essential central eIF4G722949 domain binds specifically to the IR ES, and its binding is strongly enhanced by eIF4A (33). The interaction of eIF4G w ith this IR ES may play a role analogous to that of eIF4E on capped mRNAs, that is, to recr uit the eIF4F c omplex and associated factors and to promote ribosomal att achment at a defined location on an mRNA.
The Role of IRES Transacting Factors in Initiation of Picornavirus Translation. The activ it y of a number of pic ornav ir us IR ESs is

subject to cell-t ype-specific restriction: for example, the attenPestova et al.

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Fig. 3. Schematic representation of EMCV, FMDV, and TMEV IRES domains HL, showing binding sites for PTB (thick gray line) and ITAF45 (black icosahedron), as determined by footprinting (30, 31), and for the eIF4G eIF4A complex, as determined by footprinting (31, 32) and toeprinting (26, 27, 33). The interaction of eIF4G eIF4A with the JK domain, which is essential for recruitment of the 43S complex to the initiation codon, is enhanced by PTB and ITAF45 in an IRES-specific manner, as discussed in the text.

uation of poliov ir us vac cine strains is in part because of a defect in translation in neuronal cells. Poliov ir us and rhinov ir us IR ESs mediate in itiation of translation ef ficiently in HeL a and K rebs 2 cells, and their restricted activ it y in RR L in vitro can be allev iated by deletion of the IR ES or by supplement ation of RR L by ribosomal salt wash f ractions f rom HeL a or K rebs cells (e.g., refs. 34, 35). Translation mediated by poliov ir us and rhinov ir us IR ESs thus depends on the interaction w ith these IR ESs of noncanon ical IR ES transacting factors (ITAFs) that are either absent f rom RR L or sign ificantly less abundant in RR L than in per missive cells. In early ex periments to identif y ITAFs required for pic ornav ir us IR ES function, we and others identified a 57-kDa protein that bound specifically to all pic ornav ir us IR ESs as the P TB, a cellular polypeptide that c ont ains four RNA-rec ogn ition motif -like domains (36 39). The P TB dependence of in itiation on the w ild-t ype EMCV IR ES is small (27, 40) but was sign ificantly enhanced by a single nucleotide insertion in the eIF4G-binding site or by alteration of the sequence downstream of the in itiation c odon (40). Footprinting analysis indicated that P TB binds multiple nonc ontiguous sites on the EMCV IR ES (ref. 30; Fig. 3). Taken together, these obser vations suggested a model in which binding of ITAFs such as P TB st abilizes an IR ES in an optimal c onfor mation for binding of essential factors and the 43S c omplex. Our analysis of in itiation on the related TMEV and FMDV IR ESs prov ided strong support for this hypothesis (31). TMEV (GDVII strain) and FMDV (t ype 01K) are neurotropic and epitheliotropic pic ornav ir uses, respectively. Their IR ESs are 40% identical and are closely related to the EMCV IR ES. Substitution of the IR ES in an infectious genomic TMEV clone by that of FMDV strongly attenuated the neurov ir ulence of the resulting chimeric v ir us w ithout sign ificantly af fecting its abilit y to replicate in cultured BHK-21 cells or to be translated in vitro in RR L (31). We used biochemical rec onstitution of the in itiation process on these mRNAs to define the molecular basis for this cell t ype-specific dif ference in the function of these IR ESs. In itiation on the FMDV and TMEV IR ESs had identical requirements for canon ical in itiation factors to those described above for EMCV. However, whereas in itiation on the EMCV IR ES was only weakly stimulated by P TB, in itiation on the TMEV IR ES depended strongly on P TB, and in itiation on the FMDV IR ES required both P TB and a 45kDa ITAF (ITAF45).
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We purified and sequenced ITAF45 and found that it is identical to a prev iously identified proliferation-dependent protein that is not ex pressed in nonproliferating cells such as neurons (31). The absence of this factor may ac c ount for the inabilit y of the chimeric TMEV v ir us to replicate in neurons. The activ ities of P TB and ITAF45 in promoting 48S c omplex for mation on the FMDV IR ES were strongly synergistic. These ITAFs bound to nonoverlapping sites on the IR ES (Fig. 3) and together caused localized c onfor mational changes in it, specifically in regions adjacent to the binding site for the eIF4G eIF4A c omplex. The interaction of the IR ES w ith the eIF4G eIF4A c omplex is essential for in itiation and, sign ificantly, this interaction was specifically enhanced by these t wo ITAFs. EMCV, FMDV, and TMEV IR ESs all bind to P TB and ITAF45, so it is the requirement for them rather than their abilit y to interact that dif fers as a c onsequence of sequence dif ferences bet ween these IR ESs. Similar obser vations have also been made for the sec ond group of pic ornav ir us IR ESs, members of which are also closely related to each other yet also appear to have dif ferent ITAF requirements. For example, poliov ir us and rhinov ir us IR ESs both bind to P TB, to the poly(rC)-binding protein 2 (PCBP2), and to unr (35, 41). P TB c ont ains four RNA-rec ogn ition motiflike domains, PCBP2 has three KH domains that likely c onstitute its RNA-binding sur face, and unr c ont ains five c old-shock RNA-binding domains. These polypeptides therefore all have the potential to make multipoint interactions w ith these IR ESs and to st abilize their folding in an active c onfor mation. However, whereas in itiation on the rhinov ir us IR ES depends on unr, strongly enhanced by P TB and less responsive to PCBP2, the poliov ir us IR ES depends on P TB and PCBP2 and does not respond to unr (35). Our analysis of in itiation on EMCV-like IR ESs suggests a model that w ill likely be applicable to poliov ir us-like IR ESs and possibly to some other v iral and cellular IR ESs. Specific binding of eIF4F (or its eIF4G and eIF4A subun its) to the IR ES is required to mediate internal ribosomal entr y and itself depends on the eIF4F and ribosomal binding sites hav ing the c orrect c onfor mation. The role of ITAFs is to bind an IR ES to enable it to att ain or maint ain this c onfor mation, for binding both of eIF4G eIF4A and possibly of other c omponents of the 43S c omplex. The diversit y of IR ES sequences and str uctures leads to the requirement for a variet y of ITAFs.
Internal Initiation by Factor-Independent Binding of Ribosomes to the Initiation Codon. The 5 NTRs of HCV and of the related classical

sw ine fever v ir us (CSFV) and bov ine v iral diarrhea v ir us (BVDV) also promote translation by cap-independent internal ribosomal entr y (e.g., ref. 42). IR ESs are defined solely by functional criteria and cannot yet be predicted by the presence of characteristic RNA sequence or str uctural motifs. As a r ule, there are no sign ificant similarities bet ween indiv idual IR ESs (unless they are f rom related v ir uses). The related BVDV, CSFV, and HCV IR ESs are the best characterized members of an IR ES group that is wholly distinct f rom both the EMCV-like and poliov ir us-like groups of IR ESs w ith regard to length, sequence, and str ucture. We investigated in itiation on BVDV, CSFV, and HCV IR ESs to deter mine whether all IR ESs mediate internal in itiation of translation by a single c ommon mechan ism irrespective of sequence variation and, if not, to characterize un ique aspects of in itiation on this group of HCVlike IR ESs. The BVDV, CSFV, and HCV 5 NTRs are 385, 372, and 342 nt long, respectively, and although they dif fer f rom each other at 3550% of base positions, many of these nucleotide dif ferences are c ovariant substitutions, indicative of c onser ved higher order str ucture. Even minor mut ations in str uctural elements subst antially reduced IR ES activ it y, but this c ould in most inst ances be restored by c ompensator y sec ond site mut ations that restored
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Fig. 4. Schematic secondary structure of domains II, III, and IV of HCV-like IRESs, showing sites of interaction with eIF3 (thick black lines) (47) and with 40S subunits (thick gray lines) (48, 49). The toeprints detected at the leading edge of bound eIF3 (46, 47, 49, 50) are indicated by an arrow. The toeprints at the leading edge of 40S subunits in binary IRES:40S subunit complexes are indicated by open circles and in 48S complexes formed on inclusion of eIF2 GTP Met-tRNAi with 40S subunits by filled circles (46, 50). Toeprints caused by ribosomal contact with the pseudoknot are not shown. Sequences flanking the initiation codon are base paired to form domain IV in HCV but not in BVDV and CSFV. BVDV and CSFV contain two hairpins (IIId1 and IIId2) at an analogous position to HCV IIId. The nomenclature of helices in the pseudoknot and of domains is as in ref. 48.

sec ondar y str ucture (43 46). The most highly c onser ved residues are of ten unpaired and may thus be able to interact w ith c omponents of the translation apparatus. These results and obser vations have led to a model for IR ES function in which str uctural elements in the IR ES act as a scaf fold that orients these potential binding sites in such a way that their interaction w ith factors and ribosomes leads to assembly of functional ribosomal in itiation c omplexes. These HCV-like 5 NTRs c onsist of four major str uctural domains (II V) and a c omplex pseudok notted str ucture bet ween domains II, III, and I V (Fig. 4). HCV domain I V is base-paired, whereas equivalent residues in BVDV and CSFV are not. The boundaries of these IR ESs extend f rom the 3 border of the basal helix of domain II to the in itiation c odon, and IR ES activ it y is af fected by the c oding sequence downstream of the in itiation c odon. Minor mut ations in domain II, domain III, and the pseudok not can cause subst antial reductions in IR ES activ it y. We deter mined the min imum set of factors required for assembly of 48S c omplexes on these IR ESs by in vitro rec onstitution by using purified translation c omponents (46, 50). The most remark able aspect of in itiation on these IR ESs is that they bind 40S subun its specifically and st ably, in the absence of in itiation factors, so that the ribosomal P site is placed in the immediate prox imit y of the in itiation c odon. Addition of eIF2 GT P Met-tRNAi is suf ficient for the bound 40S subun it to lock onto the in itiation c odon. The direct att achment of the 43S c omplex to the in itiation c odon is c onsistent w ith earlier reports that translation in itiation on HCV and CSFV IR ESs in RR L does not involve ribosomal scann ing af ter in itial att achment (44, 45). A lthough eIF3 is not required for assembly of this min imal 48S c omplex, eIF3 has been reported to be associated w ith f ree 40S subun its in the c y toplasm, and it is therefore likely that, in vivo, it is also a c onstituent of 48S c omplexes on these IR ESs. eIF3 itself also binds specifically and st ably to the IR ES; the independent interaction of t wo dif ferent c omponents of the 43S c omplex w ith the IR ES may enhance the af fin it y and specificit y of binding. The binding site for eIF3 has been mapped by
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toeprinting and chemical enz y matic footprinting to the apical half of domain III (ref. 47; Fig. 4) and includes subdomain IIIb and junction IIIabc. Not ably, 48S c omplex for mation on HCV-like IR ESs has no requirement for eIF4A, 4B, 4E, or 4G, nor any requirement for AT P hydrolysis. Translation mediated by these IR ESs is also not inhibited by dominant negative eIF4A mut ants (46, 50). It thus dif fers fundament ally f rom cap-mediated in itiation and in itiation mediated by pic ornav ir us IR ESs of both the EMCV and poliov ir us-like groups. The in itiation factors eIF4A, 4B, 4E, and 4G do not inf luence in itiation c omplex for mation on HCV-like IR ESs, and indeed these factors are probably unable to gain ac cess to and unw ind the region of the IR ES immediately upstream and downstream of the in itiation c odon that enters the mRNA-binding clef t of the 40S subun it. For example, translation ef ficienc y is strongly reduced by mut ations that increase base pairing in HCV domain I V (which c ont ains the in itiation c odon) and thus st abilize it (51) and by introduction of a hairpin immediately downstream of the CSFV in itiation c odon (52). Sec ondar y str uctures of equivalent st abilit y are readily unwound during cap-mediated in itiation. In itiation on prok ar yotic mRNAs involves factor-independent binding of small (30S) ribosomal subun its as a result of base pairing bet ween the linear Shine Dalgarno (SD) sequence in mRNAs and c omplement ar y linear anti-SD sequences in the ribosomal 16S rRNA (52). A lthough there are strik ing analogies bet ween this mechan ism and the factor-independent binding of 40S subun its to HCV-like IR ESs, it is ev ident that binding of 40S subun its is deter mined by multiple nonc ontiguous sequences in the IR ES rather than by a single linear sequence. We do not yet k now whether binding of an IR ES to the 40S subun it involves RNA-RNA base pairing w ith 18S rRNA. The only c ont act identified so far is w ith a ribosomal protein, but this interaction likely is not a primar y deter minant of the IR ES 40S subun it interaction (46, 48). Toeprinting and deletion analyses indicated that a 40S subun it interacts w ith the IR ES at multiple sites; primer extension is arrested by bound 40S subun its in the pseudok not and downstream of the in itiation c odon (Fig. 4). We used enz y matic footprinting to identif y the principal sites in HCV and CSFV IR ESs that are protected f rom cleavage by bound 40S subun its (48, 49). Similar interaction sites were identified in these t wo IR ESs, and they are located in regions of high sequence c onser vation in HCV-like IR ESs. These sites include the apex of HCV subdomain IIId and the equivalent CSFV subdomain IIId1, the pseudok not, and nucleotides f lank ing the in itiation c odon. The number of protected residues in the last of these sites c orresponds closely to the length estimated for the mRNAbinding clef t in 40S subun its, and it is therefore likely that additional upstream c ont acts bet ween the IR ES and the 40S subun it involve regions of the 40S subun it outside this clef t. The ribosomal binding sur face of the IR ES is therefore extensive; these footprinting and mut ational analyses (see below) suggest that it does not overlap the eIF3-binding site except in subdomain IIIa. The import ance of these sites of interaction w ith the 40S subun it for IR ES function is supported by the results of mut ational analysis. The apical residues GGG266 268 in HCV IIId and analogous residues (GGG268 270) in CSFV IIId1 are essential for ribosomal binding and IR ES function (49, 53). The pseudok not has long been k nown to be functionally import ant (43, 45, 46). We found that substitutions in its 5 helical segment abrogate ribosomal binding, whereas substitutions in its 3 helix do not prevent ribosomal att achment to the IR ES but impair binding of sequences f lank ing the in itiation c odon to the ribosomal mRNA-binding clef t (46, 49). Consistent w ith this c onclusion, we found that residues f lanking the in itiation c odon are also not required for ribosomal
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att achment to the IR ES to for m a st able binar y c omplex, even though they c onstitute a major site of interaction bet ween these IR ESs and the 40S subun it. Similarly, deletion of domain II or mut ations in domain IIIa also impaired binding of the in itiation c odon region to the ribosomal mRNA-binding clef t but did not prevent binar y c omplex for mation. These parts of the IR ES therefore do not c ont ain primar y deter minants of ribosomal binding (48, 49). We c onclude that HCV-like IR ESs c ont ain one set of deter minants that is required for in itial ribosomal att achment (including subdomain IIId IIId1, adjacent regions of domain III, and the 5 helical segment of the pseudok not) and a sec ond set of deter minants (including domain II, the 3 helical segment of the pseudok not, and downstream sequences) that is required for, or at least promotes, subsequent ac curate placement of the in itiation c odon in the ribosomal P site. The IR ES is not a st atic str ucture, and it is likely that it undergoes str uctural transitions during these t wo st ages in ribosomal binding and subsequently during subun it join ing. The mechan ism of in itiation on HCV-like IR ESs is therefore distinct f rom both cap-mediated and EMCV IR ES-mediated in itiation in hav ing no requirement for AT P or for any member of the eIF4F group of factors. HCV-like IR ESs bypass the requirement for these factors and for eIF1 and eIF1A by v irtue of their abilit y to recr uit 43S c omplexes directly to the in itiation c odon by binding specifically to eIF3 and to the 40S subun it. The import ance of the integrit y of the str ucture of these IR ESs for this mode of translation in itiation suggests that these IR ESs c onstitute valid t argets for potential chemotherapeutic agents such as antibiotics that c ould bind the IR ES and distort the str ucture of binding sites for these c omponents of the translation apparatus.
Perspectives. We have characterized the outlines of three dif fer-

1. Pestova, T. V., Bor ukhov, S. I. & Hellen, C. U. T. (1998) Nature (London) 394, 854 859. 2. Morino, S., Imat ak a, H., Sv itk in, Y. V., Pestova, T. V. & Sonenberg, N. (2000) Mol . Cell . Biol . 20, 468 477. 3. Chaudhuri, J., Chowdhur y, D. & Maitra, U. (1999) J. Biol . Chem. 274, 1797517980. 4. Battiste, J. L., Pestova, T. V., Hellen, C. U. T. & Wagner, G. (2000) Mol. Cell 5, 109 119. 5. Dahlquist, K. D. & Puglisi, J. D. (2000) J. Mol . Biol . 299, 115. 6. Yoon, H. & Donahue, T. F. (1992) Mol . Cell . Biol . 12, 248 260. 7. Fletcher, C. M., Pestova, T. V., Hellen, C. U. T. & Wagner, G. (1999) EMBO J. 18, 26312637. 8. Pestova, T. V. & Hellen, C. U. T. (1999) Trends Biochem. Sci . 24, 85 87. 9. Chakrabarti, A. & Maitra, U. (1991) J. Biol . Chem. 266, 14039 14045. 10. Asano, K., Clay ton, J., Shalev, A. & Hinnebusch, A. G. (2000) Genes Dev. 14, 2534 2546. 11. Huang, H. K., Yoon, H., Hann ig, E. M. & Donahue, T. F. (1997) Genes Dev. 11, 2396 2413. 12. Pestova, T. V., Hellen, C. U. T. & Dever, T. E. (2000) in Translational Control of Gene Expression, eds. Sonenberg, N., Mathews, M. B. & Hershey, J. W. B. (Cold Spring Harbor L ab. Press, Plainv iew, NY), pp. 425 445.

13. Pestova, T. V., L omak in, I. B., Lee, J. H., Choi, S. K., Dever, T. E. & Hellen, C. U. T. (2000) Nature (London) 403, 332335. 14. Choi, S. K., Lee, J. H., Zoll, W. L., Merrick, W. C. & Dever, T. E. (1998) Science 280, 17571760. 15. Lee, J. H., Choi, S. K., Roll-Mecak, A., Burley, S. K. & Dever, T. E. (1999) P roc. Natl . Acad . Sci . USA 96, 1066 1070. 16. Carrera P., Johnstone, O., Nak amura, A., Casanova, J., Jackle, H. & L ask o P. (2000) Mol . Cell 5, 181187. 17. Kolak ofsky, D., Oht a, T. & Thach, R. E. (1968) Nature (London) 220, 244 247. 18. Jang, S.-K., K rЁusslich, H.-G., Nicklin, M. J. H., Duke, G. M., Palmenberg, a A. C. & Wimmer, E. (1988) J. V irol . 62, 2636 2643. 19. Pelletier, J. & Sonenberg, N. (1988) Nature (London) 334, 320 325. 20. Pilipenk o, E. V., Blinov, V. M., Chernov, B. K., Dmitrieva, T. M. & Agol, V. I. (1989) Nucleic Acids Res. 17, 57015711. 21. Pilipenk o, E. V., Blinov, V. M., Romanova, L. I., Sinyak ov, A. N., Maslova, S. V. & Agol, V. I. (1989) V irolog y 168, 201209. 22. Kaminsk i, A., Howell, M. T. & Jackson, R. J. (1990) EMBO J. 9, 37533759. 23. Pilipenk o, E. V., Gmyl, A. P., Maslova, S. V., Belov, G. A., Sinyak ov, A. N., Huang, M., Brown, T. D. K. & Agol, V. I. (1994) J. Mol . Biol . 241, 398 414. 24. Hellen, C. U. T., Pestova, T. V. & Wimmer, E. (1994) J. V irol . 68, 6312 6322.

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ent mechan isms of translation in itiation by using biochemical rec onstitution to deter mine the min imum set of factors required for assembly of 48S and 80S ribosomal c omplexes on three distinct t ypes of euk ar yotic mRNA and by using toeprinting and footprinting to map the location of translation c omponents on these mRNAs. The findings reported here raise both general and specific questions about translation in itiation. The finding that internal ribosomal entr y on the t wo t ypes of IR ES that we have examined oc curs by ver y dif ferent mechan isms indicates there is no single mode of internal ribosomal entr y. Indeed, the implicit possibilit y that there are yet other mechan isms for in itiation directed by an IR ES has been borne out by recent analysis of in itiation on the intergen ic IR ES of cricket paralysis v ir us (CrPV), which remark ably requires nei-

ther in itiator tRNA nor in itiation factors (54). Like HCV and related IR ESs, this CrPV IR ES also binds directly to 40S subun its but in a sign ificantly dif ferent manner, such that the P site is apparently filled by a pseudok not and is inac cessible to the eIF2 GT P Met-tRNA i c omplex. Because the number of k nown cellular and v iral IR ESs is c onst antly grow ing, we cannot r ule out that additional mechan isms of internal ribosomal entr y ex ist that are distinct f rom those used by EMCV, HCV, or CrPV-like IR ESs. It seems probable that even those IR ESs on which in itiation oc curs by a mechan ism fundament ally similar to one of these three groups of IR ESs w ill nevertheless require ITAFs dif ferent f rom those identified to date. It w ill be interesting to see whether the ``induced active c onfor mation'' model for ITAF function described for the FMDV IR ES (31) w ill be more generally applicable. Just as it is unlikely that in itiation on all IR ESs w ill be described by one of the three models described above, so it would be premature to assume that in itiation on all capped mRNAs oc curs by the mechan ism that we have described for -globin mRNA. More specifically, our k nowledge of the scann ing process is ver y r udiment ar y, and a number of open questions need to be addressed in the near future. These questions include: (i) What are the molecular interactions and c onfor mational changes that lead to binding of a 43S c omplex to the capped eIF4F-bound 5 end of an mRNA? (ii) How and when are interactions bet ween cap-bound factors and the 43S c omplex dissociated as this c omplex begins to scan f rom the cap-prox imal region of an mRNA? (iii) Is ribosomal movement on the 5 NTR obligatorily linked to ``melting'' sec ondar y str ucture in the 5 NTR, and, if these processes can be unc oupled, is the 43S c omplex intrinsically capable of movement on mRNA w ithout c onc omit ant AT P hydrolysis? (iv) Which factors inf luence the processiv it y of scann ing? (v) How does the local sequence c ontext of an in itiation c odon inf luence the ef ficienc y of in itiation at that c odon? (vi) How does rec ogn ition of the in itiation c odon trigger all of the events associated w ith subun it join ing? A nswers to these questions not only w ill lead to a more det ailed underst anding of the molecular mechan ism of the in itiation process but also w ill of fer insights into how str uctural dif ferences bet ween dif ferent mRNAs deter mine when and how ef ficiently they are translated.
Research done in our laboratories was supported Grants A I44108 01 and GM59660 f rom the National Institutes of Health (to C.U.T.H. and T.V.P.), by Grant MCB-9726958 f rom the National Science Foundation (to C.U.T.H.), and by grants f rom the Council for Tobac c o Research Council (to C.U.T.H.), the Howard Hughes Medical Institute (to I.N.S. and C.U.T.H.), and the Russian Foundation of Basic Research (to V.I.A. and I.N.S.).

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40. Kaminsk i, A. & Jackson, R. J. (1998) RNA 4, 626 638. 41. Walter, B. L., Nguyen, J. H., Ehrenfeld, E. & Semler, B. L. (1999) RNA 5, 1570 1585. 42. Tsuk iyama-Kohara, K., Iizuk a, N., Kohara, M. & Nomoto, A. (1992) J. V irol . 66, 1476 1483. 43. Wang, C., Le, S. Y., A li, N. & Siddiqui, A. (1995) RNA 1, 526 537. 44. Reynolds, J. E., Kaminsk i, A., Carroll, A. R., Clarke, B. E., Rowlands, D. J. & Jackson, R. J. (1996) RNA 2, 867 878. 45. Rijnbrand, R., van der Straaten, T., van Rijn, P. A., Spaan, W. J. M. & Bredenbeek, P. J. (1997) J. V irol . 71, 451 457. 46. Pestova, T. V., Shatsky, I. N., Fletcher, S. P., Jackson, R. J. & Hellen, C. U. T. (1998) Genes Dev. 12, 67 83. 47. Sizova, D.V., Kolupaeva, V. G., Pestova, T. V., Shatsky, I. N. & Hellen, C. U. T. (1998) J. V irol . 72, 4775 4782. 48. Kolupaeva, V. G., Pestova, T. V. & Hellen, C. U. T. (2000) J. V irol . 74, 6242 6250. 49. Kolupaeva, V. G., Pestova, T. V. & Hellen, C. U. T. (2000) RNA 6, 17911807. 50. Pestova, T. V. & Hellen, C. U. T. (1999) V irolog y 258, 249 256. 51. Honda, M., Brown, E. A. & Lemon, S. M. (1996) RNA 2, 955968. 52. Jackson, R. J. (2000) in Translational Control of Gene Expression, eds. Sonenberg, N., Mathews, M. B. & Hershey, J. W. B. (Cold Spring Harbor L ab. Press, Plainv iew, NY), pp. 127183. 53. Jubin, R., Vantuno, N. E., K ief t, J. S., Murray, M. G., Doudna, J. A., L au, J. Y. & Baroudy, B. M. (2000) J. V irol . 74, 10430 10437. 54. Wilson, J. E., Pestova, T. V., Hellen, C. U. T. & Sarnow, P. (2000) Cell 102, 511520.

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