The mechanism of ribosome binding to eucaryotic mRNAs is not well understood, but it requires the participation of eucaryotic initiation factors eIF-4A, eIF-4B, and eIF-4F and the hydrolysis of ATP. Evidence has accumulated in support of a model in which these initiation factors function to unwind the 5'-proximal secondary structure in mRNA to facilitate ribosome binding. To obtain direct evidence for initiation factor-mediated RNA unwinding, we developed a simple assay to determine RNA helicase activity, and we show that eIF-4A or eIF-4F, in combination with eIF-4B, exhibits helicase activity. A striking and unprecedented feature of this activity is that it functions in a bidirectional manner. Thus, unwinding can occur either in the 5'-to-3' or 3'-to-5' direction. Unwinding in the 5'-to-3' direction by eIF-4F (the cap-binding protein complex), in conjunction with eIF-4B, was stimulated by the presence of the RNA 5' cap structure, whereas unwinding in the 3'-to-5' direction was completely cap independent. These results are discussed with respect to cap-dependent versus cap-independent mechanisms of ribosome binding to eucaryotic mRNAs.A critical step in eucaryotic protein biosynthesis is binding of the small (40S) ribosomal subunit to mRNA (36,39). This step is rate limiting in translation initiation (25) and is a key target for regulation (reviewed in reference 53), but the mechanism of this process is poorly understood. Two pathways for the binding of 40S ribosomal subunits to mRNA have been described, which differ in their requirement for the cap structure. The 5' cap structure, m7GpppX (where X is any nucleotide) is a nearly ubiquitous feature of all eucaryotic mRNAs (49). Evidence indicates that translation initiation of the majority of eucaryotic mRNAs is accomplished in a cap-enhanced manner, whereby 40S ribosomal binding to mRNA is facilitated by the cap structure (reviewed in references 3 and 50). Recently, it has been shown that poliovirus (42) and encephalomyocarditis virus (26) mRNAs, which are naturally uncapped (13,21,38), initiate translation by a different mechanism. In this case, the 40S subunit binds directly to an internal element on the picornavirus 5' untranslated region, by-passing upstream sequences and the requirement for the cap structure.Although the two initiation pathways are mechanistically distinguishable, they nevertheless require a similar set of initiation factors (eIFs [57]) to bind 40S ribosomal subunits to mRNA. Cap-stimulated mRNA binding to the small ribosomal subunit requires at least three initiation factors, eIF-4A, eIF-4B, and eIF-4F, in addition to the hydrolysis of ATP (reviewed in references 10, 44, and 53). eIF-4F is a multisubunit complex consisting of three major polypeptides of 24, 50, and 220 kilodaltons (kDa) (8,20,59). The 24-kDa polypeptide is the cap-binding subunit, which also exists in a free form, termed eIF-4E (55). The 50-kDa polypeptide is a structural variant of free eIF-4A (8,20). Although eIF-4F contains an eIF-4A subunit which is almost identical to...

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2α (eIF2α). Vaccinia virus E3Lencodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2α, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and λ repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.

To understand how phosphorylation of eukaryotic translation initiation factor (eIF)-2␣ in Saccharomyces cerevisiae stimulates GCN4 mRNA translation while at the same time inhibiting general translation initiation, we examined the effects of altering the gene dosage of initiator tRNA Met , eIF-2, and the guanine nucleotide exchange factor for eIF-2, eIF-2B. Overexpression of all three subunits of eIF-2 or all five subunits of eIF-2B suppressed the effects of eIF-2␣ hyperphosphorylation on both GCN4-specific and general translation initiation. Consistent with eIF-2 functioning in translation as part of a ternary complex composed of eIF-2, GTP, and Met-tRNA i Met , reduced gene dosage of initiator tRNA Met mimicked phosphorylation of eIF-2␣ and stimulated GCN4 translation. In addition, overexpression of a combination of eIF-2 and tRNA i Met suppressed the growthinhibitory effects of eIF-2 hyperphosphorylation more effectively than an increase in the level of either component of the ternary complex alone. These results provide in vivo evidence that phosphorylation of eIF-2␣ reduces the activities of both eIF-2 and eIF-2B and that the eIF-2 ⅐ GTP ⅐ Met-tRNA i Met ternary complex is the principal component limiting translation in cells when eIF-2␣ is phosphorylated on serine 51. Analysis of eIF-2␣ phosphorylation in the eIF-2-overexpressing strain also provides in vivo evidence that phosphorylated eIF-2 acts as a competitive inhibitor of eIF-2B rather than forming an excessively stable inactive complex. Finally, our results demonstrate that the concentration of eIF-2 ⅐ GTP ⅐ Met-tRNA i Met ternary complexes is the cardinal parameter determining the site of reinitiation on GCN4 mRNA and support the idea that reinitiation at GCN4 is inversely related to the concentration of ternary complexes in the cell.The current model for the mechanism of translation initiation in eukaryotic cells derives from biochemical analysis of mammalian cell-free systems and characterization of individual reactions with purified initiation factors. These studies have identified eukaryotic initiation factor (eIF)-2 as the protein responsible for binding the initiator Met-tRNA (MettRNA i Met ) to the 40S ribosomal subunit in an early step of the initiation pathway (see reviews in references 26 and 31). It is believed that the Met-tRNA i Met is delivered as part of a ternary complex composed of eIF-2, GTP, and Met-tRNA i Met . After binding of the ternary complex to the 40S ribosomal subunit, the GTP is hydrolyzed to GDP and eIF-2 is released in a binary complex with GDP. Because mammalian eIF-2 has a 100-to 400-fold-higher affinity for GDP than for GTP, a guanine nucleotide exchange factor known as eIF-2B is required to recycle eIF-2 ⅐ GDP back to eIF-2 ⅐ GTP, allowing eIF-2 to function in a subsequent round of translation initiation (26,36). Phosphorylation of eIF-2␣ on serine 51 inhibits the exchange of GTP for GDP on eIF-2. Not only is exchange blocked on the phosphorylated eIF-2 molecule, but phosphorylated eIF-2 also prevents eIF-2B from re...

In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae. The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs.