We have examined the role of the mammalian initiation factor eIF1 in the formation of the 40 S preinitiation complex using in vitro binding of initiator Met-tRNA (as Met-tRNA i ⅐eIF2⅐GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA. We observed that, although both eIF1A and eIF3 are essential to generate a stable 40 S preinitiation complex, quantitative binding of the ternary complex to 40 S subunits also required eIF1. The 40 S preinitiation complex contained, in addition to eIF3, both eIF1 and eIF1A in a 1:1 stoichiometry with respect to the bound Met-tRNA i . These three initiation factors also bind to free 40 S subunits, and the resulting complex can act as an acceptor of the ternary complex to form the 40 S preinitiation complex (40 S⅐eIF3⅐eIF1⅐eIF1A⅐Met-tRNA i ⅐eIF2⅐GTP). The stable association of eIF1 with 40 S subunits required the presence of eIF3. In contrast, the binding of eIF1A to free 40 S ribosomes as well as to the 40 S preinitiation complex was stabilized by the presence of both eIF1 and eIF3. These studies suggest that it is possible for eIF1 and eIF1A to bind the 40 S preinitiation complex prior to mRNA binding.The initiation of translation in eukaryotic cells occurs by a sequence of partial reactions that require a number of specific proteins called eukaryotic (translation) initiation factors (eIFs).1 According to the currently accepted view of translation initiation, primarily derived from in vitro studies with purified initiation factors, an obligatory intermediate step in the overall initiation reaction is the binding of the initiator Met-tRNA i as the Met-tRNA i ⅐eIF2⅐GTP ternary complex to a 40 S ribosomal subunit containing bound initiation factor eIF3. This interaction leads to the production of the 40 S preinitiation complex (40 S⅐eIF3⅐Met-tRNA i ⅐eIF2⅐GTP). The 40 S preinitiation complex then binds to the 5Ј-capped end of mRNA and scans the mRNA in a 5Ј33Ј direction until the 40 S complex encounters the initiating AUG codon to form the 40 S initiation complex (40 S⅐eIF3⅐mRNA⅐Met-tRNA i ⅐eIF2⅐GTP). This reaction requires the participation of three other initiation factors eIF4F, eIF4A, and eIF4B. Subsequently, the 60 S ribosomal subunit joins the 40 S complex in a reaction dependent on two other factors, eIF5 and eIF5B, to form a functional 80 S initiation complex (80 S⅐mRNA⅐Met-tRNA i ) (for review, see Refs. 1-5).In addition to the initiation factors described above, the 17-kDa eIF1A and the 12-kDa eIF1 are also known to play essential roles in the overall initiation process (1-5). Earlier biochemical studies demonstrated that both eIF1 and eIF1A have a weak stimulatory effect on the binding of Met-tRNA i and mRNA to 40 S and 80 S initiation complexes in the presence of other factors (6 -11). The presence of eIF1A in the 40 S initiation complex was also shown in one of these earlier studies (9). In vivo studies in Saccharomyces cerevisiae demonstrated that the genes encoding these two small initiation factors are essential for initiation of protein synthesis an...

Genetic studies in yeast have shown that the translation initiation factor eIF5 plays an important role in the selection of the AUG start codon. In order to ensure translation fidelity, the hydrolysis of GTP bound to the 40S preinitiation complex (40S . Met-tRNA i . eIF2 . GTP), promoted by eIF5, must occur only when the complex has selected the AUG start codon. However, the mechanism that prevents the eIF5-promoted GTP hydrolysis, prior to AUG selection by the ribosomal machinery, is not known. In this work, we show that the presence of initiation factors eIF1, eIF1A and eIF3 in the 40S preinitiation complex (40S . eIF1 . eIF1A . eIF3 . Met-tRNA i . eIF2 . GTP) and the subsequent binding of the preinitiation complex to eIF4F bound at the 5 0 -cap structure of mRNA are necessary for preventing eIF5-promoted hydrolysis of GTP in the 40S preinitiation complex. This block in GTP hydrolysis is released upon AUG selection by the 40S preinitiation complex. These results, taken together, demonstrate the biochemical requirements for regulation of GTP hydrolysis and its coupling to the AUG selection process during translation initiation.

The biosynthesis of 60 S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eIF6. This protein is necessary for the formation of 60 S ribosomal subunits because it is essential for the processing of 35 S pre-rRNA to the mature 25 S and 5.8 S rRNAs. In the present work, using molecular genetic and biochemical analyses, we show that Hrr25p, an isoform of yeast casein kinase I, phosphorylates Tif6p both in vitro and in vivo. Tryptic phosphopeptide mapping of in vitro phosphorylated Tif6p by Hrr25p and 32 Plabeled Tif6p isolated from yeast cells followed by mass spectrometric analysis revealed that phosphorylation occurred on a single tryptic peptide at Ser-174. Sucrose gradient fractionation and coimmunoprecipitation experiments demonstrate that a small but significant fraction of Hrr25p is bound to 66 S preribosomal particles that also contain bound Tif6p. Depletion of Hrr25p from a conditional yeast mutant that fails to phosphorylate Tif6p was unable to process pre-rRNAs efficiently, resulting in significant reduction in the formation of 25 S rRNA. These results along with our previous observations that phosphorylatable Ser-174 is required for yeast cell growth and viability, suggest that Hrr25p-mediated phosphorylation of Tif6p plays a critical role in the biogenesis of 60 S ribosomal subunits in yeast cells. Eukaryotic translation initiation factor 6 (eIF6)3 was initially purified as a protein that can bind the 60 S ribosomal subunit and prevent its association with the 40 S ribosomal subunit (1-4). Based on this ribosomal subunit anti-association property, the protein was originally thought to be an initiation factor that functions to provide a pool of free ribosomal subunits required for initiation of protein synthesis (5). The protein was named eIF6, although a role in translation was not demonstrated in these earlier studies. To understand the function of this protein in translation, Si et al. (6) first cloned the human cDNA and then the yeast Saccharomyces cerevisiae gene (7) encoding functionally active eIF6, each of 245 amino acids. The two proteins are 72% identical. The yeast gene, designated TIF6, is a single copy gene that is essential for cell growth and viability (7). These properties of TIF6 allowed the construction of a conditional null allele by placing its expression under the control of the regulatable GAL10 promoter. Depletion of Tif6p in this yeast mutant strain inhibited the rate of in vivo protein synthesis (7). However, a more detailed analysis of the protein synthesis parameters in Tif6p-depleted cells showed that the reduced rate of protein synthesis was not due to a direct inhibition in initiation (7). Rather, the biogenesis of 60 S ribosomal subunits was severely inhibited. Similar observations were also reported by Sanvito et al. (8), who identified eIF6 from mammalian cells as a ␤4 integrin-interacting protein. Specifically, lack of Tif6p in yeast cells prevented the processing of pre-ribosomal RNA (pre-rRNA) to the mature 25 S and 5.8 S...