PREFACE Protein synthesis is principally regulated at the initiation stage (rather than during elongation or termination), allowing rapid, reversible and spatial control over gene expression. Progress over recent years in determining the structures and activities of initiation factors, and in mapping their interactions within ribosomal initiation complexes, has significantly advanced our understanding of the complex translation initiation process. These developments have provided a solid foundation for studies of regulation of initiation by mechanisms that include modulation of the activity of initiation factors (which affects almost all scanning-dependent initiation), or via sequence-specific RNA-binding proteins and microRNAs (which thus impact individual mRNAs).

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Initiation of translation of hepatitis C virus and classical swine fever virus mRNAs results from internal ribosomal entry. We reconstituted internal ribosomal entry in vitro from purified translation components and monitored assembly of 48S ribosomal preinitiation complexes by toe-printing. Ribosomal subunits (40S) formed stable binary complexes on both mRNAs. The complex structure of these RNAs determined the correct positioning of the initiation codon in the ribosomal ''P'' site in binary complexes. Ribosomal binding and positioning on these mRNAs did not require the initiation factors eIF3, eIF4A, eIF4B, and eIF4F and translation of these mRNAs was not inhibited by a trans-dominant eIF4A mutant. Addition of Met-tRNA i Met , eIF2, and GTP to these binary ribosomal complexes resulted in formation of 48S preinitiation complexes. The striking similarities between this eukaryotic initiation mechanism and the mechanism of translation initiation in prokaryotes are discussed. Protein synthesis begins following assembly of an initiation complex in which the initiation codon of an mRNA and the anticodon of initiator tRNA are base-paired in the ribosomal ''P'' site. There are similarities and significant differences in the mechanisms of initiation complex formation in prokaryotes and eukaryotes. A universal characteristic is that initiation starts with separated ribosomal subunits.In prokaryotes, the small (30S) ribosomal subunit binds mRNA and initiator tRNA in random order to form a complex that then undergoes conformational rearrangement, promoting codon-anticodon base-pairing at the P site and joining of the large (50S) subunit (Gualerzi and Pon 1990). Ribosome binding results from interactions between the 30S subunit and multiple recognition elements in the mRNA, such as the Shine-Dalgarno sequence (McCarthy and Brimacombe 1994). It does not depend on initiation factors. Ribosome-binding sites can occur at any position within an mRNA and as a result many prokaryotic mRNAs are polycistronic.In contrast, the small (40S) ribosomal subunit in eukaryotes requires several eukaryotic initiation factors (eIFs), first to bind initiator tRNA (as a ternary complex with eIF2 and GTP) to form a 43S complex and then to bind mRNA to form a 48S complex (Merrick 1992). The most common mechanism for recruitment of an mRNA is mediated by its capped 5Ј end, which is bound by the eIF4E subunit of eIF4F and then by the 43S complex. Ribosomal binding to mRNA and scanning to the initiation codon require ATP hydrolysis and involve eIF4A, eIF4B, and eIF4F. Most mRNAs that use this mechanism of ribosomal binding are monocistronic because initiation is usually limited to the 5Ј-most AUG codon.

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, eIF2͞GTP͞Met-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 eIF4A͞4G. 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.T ranslation of mRNA into protein begins after assembly of initiator tRNA (Met-tRNA i ), mRNA, and separated 40S and 60S ribosomal subunits into an 80S ribosome in which MettRNA i is positioned in the ribosomal P site at the initiation codon. The complex initiation process that leads to 80S ribosome formation consists of several linked stages that are mediated by eukaryotic initiation factors. These stages are:(i) Selection of initiator tRNA from the pool of elongator tRNAs by eukaryotic initiation factor (eIF)2 and binding of an eIF2͞GTP͞Met-tRNA i ternary complex and other eIFs to the 40S subunit to form a 43S preinitiation complex.(ii) Binding of the 43S complex to mRNA, which in most instances occurs by a mechanism that involves initial recognition of the m 7 G cap at the mRNA 5Ј-terminus by the eIF4E (cap-binding) subunit of eIF4F. Ribosomes bind to a subset of cellular and viral mRNAs as a result of cap-and endindependent internal ribosomal entry.(iii) Movement of the mRNA-bound ribosomal complex along the 5Ј nontranslated region (5ЈNTR) from its initial binding site to the initiation codon to form a 48S initiation complex in which the initiation codon is base paired to the anticodon of initiator tRNA.(iv) Displacement of factors from the 48S complex and joining of the 60S subunit to form an 80S ribosome, leaving Met-tRNA i in the ribosomal P site.Research in ...