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attachment to the IRES to form a stable binary complex, even though they constitute a major site of interaction between these IRESs and the 40S subunit. Similarly, deletion of domain II or mutations in domain IIIa also impaired binding of the initiation codon region to the ribosomal mRNA-binding cleft but did not prevent binary complex formation. These parts of the IRES therefore do not contain primary determinants of ribosomal binding (48, 49). We conclude that HCV-like IRESs contain one set of determinants that is required for initial ribosomal attachment (including subdomain IIId/IIId1, adjacent regions of domain III, and the 5' helical segment of the pseudoknot) and a second set of determinants (including domain II, the 3' helical segment of the pseudoknot, and downstream sequences) that is required for, or at least promotes, subsequent accurate placement of the initiation codon in the ribosomal P site. The IRES is not a static structure, and it is likely that it undergoes structural transitions during these two stages in ribosomal binding and subsequently during subunit joining.

The mechanism of initiation on HCV-like IRESs is therefore distinct from both cap-mediated and EMCV IRES-mediated initiation in having no requirement for ATP or for any member of the eIF4F group of factors. HCV-like IRESs bypass the requirement for these factors and for eIF1 and eIF1A by virtue of their ability to recruit 43S complexes directly to the initiation codon by binding specifically to eIF3 and to the 40S subunit. The importance of the integrity of the structure of these IRESs for this mode of translation initiation suggests that these IRESs constitute valid targets for potential chemotherapeutic agents such as antibiotics that could bind the IRES and distort the structure of binding sites for these components of the translation apparatus.

Perspectives. We have characterized the outlines of three different mechanisms of translation initiation by using biochemical reconstitution to determine the minimum set of factors required for assembly of 48S and 80S ribosomal complexes on three distinct types of eukaryotic mRNA and by using toeprinting and footprinting to map the location of translation components on these mRNAs. The findings reported here raise both general and specific questions about translation initiation.

The finding that internal ribosomal entry on the two types of IRES that we have examined occurs by very different mechanisms indicates there is no single mode of internal ribosomal entry. Indeed, the implicit possibility that there are yet other mechanisms for initiation directed by an IRES has been borne out by recent analysis of initiation on the intergenic IRES of cricket paralysis virus (CrPV), which remarkably requires neither initiator tRNA nor initiation factors (54). Like HCV and related IRESs, this CrPV IRES also binds directly to 40S subunits but in a significantly different manner, such that the P site is apparently filled by a pseudoknot and is inaccessible to the eIF2/GTP/Met-tRNAi complex. Because the number of known cellular and viral IRESs is constantly growing, we cannot rule out that additional mechanisms of internal ribosomal entry exist that are distinct from those used by EMCV, HCV, or CrPV-like IRESs. It seems probable that even those IRESs on which initiation occurs by a mechanism fundamentally similar to one of these three groups of IRESs will nevertheless require ITAFs different from those identified to date. It will be interesting to see whether the “induced active conformation” model for ITAF function described for the FMDV IRES (31) will be more generally applicable.

Just as it is unlikely that initiation on all IRESs will be described by one of the three models described above, so it would be premature to assume that initiation on all capped mRNAs occurs by the mechanism that we have described for ß-globin mRNA. More specifically, our knowledge of the scanning process is very rudimentary, and a number of open questions need to be addressed in the near future. These questions include: (i) What are the molecular interactions and conformational changes that lead to binding of a 43S complex to the capped eIF4F-bound 5' end of an mRNA? (ii) How and when are interactions between cap-bound factors and the 43S complex dissociated as this complex begins to scan from the cap-proximal region of an mRNA? (iii) Is ribosomal movement on the 5' NTR obligatorily linked to “melting” secondary structure in the 5' NTR, and, if these processes can be uncoupled, is the 43S complex intrinsically capable of movement on mRNA without concomitant ATP hydrolysis? (iv) Which factors influence the processivity of scanning? (v) How does the local sequence context of an initiation codon influence the efficiency of initiation at that codon? (vi) How does recognition of the initiation codon trigger all of the events associated with subunit joining? Answers to these questions not only will lead to a more detailed understanding of the molecular mechanism of the initiation process but also will offer insights into how structural differences between different mRNAs determine when and how efficiently they are translated.

Research done in our laboratories was supported Grants AI44108–01 and GM59660 from the National Institutes of Health (to C.U.T.H. and T.V.P.), by Grant MCB-9726958 from the National Science Foundation (to C.U.T.H.), and by grants from the Council for Tobacco 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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