Influence of translation factor activities on start site selection in six different mRNAs.

Current literature using biochemical assays, structural analyses and genetic manipulations has reported that the key factors associated with the faithful matching of the initiator met-tRNA to the start codon AUG are eIF1, eIF1A and eIF5. However, these findings were in each case based upon the utilization of a single mRNA, perhaps with variations. In an effort to evaluate this general finding, we tested six different mRNAs. Our results confirm that these three proteins are important for start site selection. However, two additional findings would not have been predicted. The first is that eIF1 plays a major role in selecting against start codons that are in close proximity to the 5' end of the mRNA (i.e., less than 21 nucleotides). Second, the addition of eIF5B had nearly the same affect as the addition of eIF5. This is unexpected given the different roles that eIF5 and eIF5B have been proposed to play in the 80S initiation pathway. Finally, although many of the mRNAs appear to respond qualitatively in a similar manner, the quantitative differences noted suggest that there is still some mRNA specific character to our findings. This character may be the length of the 5' UTR, involvement of an IRES element, secondary structure either 5' or 3' of the start codon or specific sequence/structure elements that interact with RNA binding proteins or the ribosome.

Characterization of mammalian eIF2A and identification of the yeast homolog.

To begin the physical characterization of eukaryotic initiation factor (eIF) 2A, a translation initiation factor that binds Met-tRNA(i), tryptic peptides from rabbit reticulocyte eIF2A were analyzed to obtain amino acid sequence information. Sequences for 8 peptides were matched to three different expressed sequence tag clones. The sequence predicted for eIF2A is 585 amino acids. Matching of the cDNA sequence to the human genome revealed that the eIF2A mRNA is made up of 15 or 16 exons, and the gene is contained on chromosome 3. A homolog in Saccharomyces cerevisiae was identified, YGR054W, which is a non-essential gene. Hemagglutinin-tagged yeast eIF2A localizes on both 40 S and 80 S ribosomes. A knockout of both eIF2A and eIF5B yielded a "synthetically sick" yeast strain with a severe slow growth phenotype. The phenotype of this double mutant and the biochemical localization suggest that eIF2A participates in translation initiation. eIF2A does not appear to participate in re-initiation as the DeltaeIF2A strain shows the same level of GCN4 induction with amino acid starvation as seen in wild type yeast. The lack of any apparent phenotype in the DeltaeIF2A strain suggests that eIF2A functions in a minor pathway, perhaps internal initiation or in the translation of a small number of specific mRNAs.