Unleashing yeast genetics on a factor-independent mechanism of internal translation initiation.

T control of gene expression is generally exerted in the initiation phase of protein synthesis. The reactions of the initiation pathway bring the ribosome to the mRNA and place the initiation codon in the peptidyl (P) site of the ribosome, base-paired with the anticodon of methionyl initiator tRNA (MettRNAi ). The aminoacyl (A) site in the ribosome is left open to accept the elongator tRNA that decodes the second codon in the ORF. Formation of this 80S initiation complex in eukaryotic cells depends on 11 or more soluble initiation factors (eIFs), identified primarily through biochemical studies of translation in rabbit reticulocyte extracts. Most of these eIFs are essential for normal cell growth in budding yeast, confirming their importance in vivo (1). Nevertheless, a number of mRNAs contain specialized regulatory elements that allow them to dispense with one or more eIFs during translation initiation. These elements, called internal ribosome entry sites (IRESs), recruit the ribosome directly to the initiation region of the mRNA. Their independence of certain eIFs allows viral mRNAs containing IRESs to be translated in infected cells where one or more eIFs have been impaired, either by the virus to provide a selective advantage for its mRNAs or by the host to impede virus gene expression. The most striking instance of this strategy is provided by cricket paralysis virus (CrPV) mRNA, which seems to be translated without any eIFs or even Met-tRNAi Met (2). The work reported by Sarnow and colleagues (3) in this issue of PNAS shows that the unique CrPV initiation mechanism can operate in budding yeast cells, so long as the canonical initiation pathway involving initiation factor 2 (eIF2) has been impaired. These findings open up the possibility of using powerful yeast genetics to dissect the details of this remarkable mechanism and to define the factors that modulate its efficiency in living cells. For most eukaryotic mRNAs, it is thought that translation initiation occurs by the scanning mechanism. In the current model for this complex process, a small (40S) ribosomal subunit carrying eIF3 and eIF1A interacts with a ternary complex (TC) consisting of eIF2 bound to GTP and Met-tRNAi . The resulting 43S complex binds to the 5 terminal m7GpppN cap structure (forming the 48S complex) and migrates into the 5 untranslated region (UTR) of the mRNA until encountering an AUG start codon in a suitable sequence context. Binding of the 43S complex to the mRNA is stimulated by the eIF4F complex, consisting of the capbinding protein eIF4E, a DEAD-box RNA helicase (eIF4A), and a scaffolding polypeptide (eIF4G) with binding sites for the other two eIFs in the 4F complex. The ATP-dependent helicase activity of eIF4A is thought to help unwind secondary structure in the 5 UTR to facilitate movement of the ribosome along the mRNA during scanning (1, 4). The poly(A)-binding protein (PABP), bound to the poly(A) tail, also interacts with eIF4G, so that both ends of the mRNA are tethered to eIF4G (5). It is thought that eIF3 bridges the interaction between this messenger ribonucleoprotein (mRNP) complex and the ribosome by interacting simultaneously with eIF4G and the 40S subunit (6). Formation of a 48S complex with MettRNAi Met base-paired to the start codon also requires eIF1 and eIF1A (7). This assembly is then recognized by eIF5, which stimulates hydrolysis of GTP in the TC, leading to release of eIF2-GDP but leaving behind Met-tRNAi Met in the P site (1, 4). Finally, eIF5B catalyzes joining of the 60S subunit to form the 80S initiation complex, with hydrolysis of a second molecule of GTP (8). An increasing number of mRNAs have been identified that are translated by alternative mechanisms, known collectively as internal initiation, in which the 40S ribosome bypasses the cap and binds to an IRES located in the 5 UTR. The best-studied IRESs, which occur in viral mRNAs, are large highly structured elements, often stabilized by RNA binding proteins. In bypassing the cap, IRESs dispense with the requirement for eIF4E, the cap-binding subunit of eIF4G (2). This fact underlies the ability of picornaviruses, including poliovirus and foot and mouth disease virus, to inhibit host cell protein synthesis by expressing proteases that cleave the PABPand eIF4E-binding domain from the N terminus of eIF4G (6, 9). It also explains how these mRNAs can be efficiently translated despite their highly structured 5 UTRs and the presence of multiple AUG codons (10) that should waylay scanning ribosomes and prevent them from reaching the authentic start site downstream (11, 12). Insertion of an IRES between the two cistrons of a dicistronic mRNA allows the downstream cistron to be translated even when capdependent translation of the first cistron is impaired, as in poliovirus-infected cells (13). Except for eIF4E, PABP, and the N terminus of eIF4G, all other canonical eIFs are required to form a 48S complex on the IRES of encephalomyocarditis virus (EMCV) (14), another picornavirus. The essential core domain of eIF4G, containing the binding sites for eIF3 and eIF4A, interacts directly with a portion of the IRES in a manner facilitated by eIF4A. This direct eIF4G–IRES interaction supplements the mRNA binding functions of eIF4E and PABP (15). Hepatitis C virus (HCV) mRNA presents a more extreme case of an IRES that functions independently of the canonical eIFs. In this case, a naked 40S ribosome can bind directly to the IRES, placing the start codon in proximity to the P site, without assistance from any other eIFs or ATP hydrolysis. Binding of Met-tRNAi Met to the start codon positioned in the P site, mediated by the TC, is still required to form an 80S initiation complex on the HCV IRES (2, 16). Recent exciting results using cryoelectron microscopy show that the 40S subunit makes numerous contacts with the IRES and undergoes a conformational change that might clamp the IRES onto the ribosome (17). The ultimate factor-independent IRESs occur in a group of insect viruses