Abstract
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.