Regulation of GTP hydrolysis during translation initiation in eukaryotes

Abstract

In eukaryotes, AUG is the most preferred codon for translation initiation. This is in contrast to prokaryotes where non-AUG codons such as GUG or UUG codons can also be used for translation initiation. How this stringency of selection of the AUG initiation codon is achieved in eukaryotes is, however, not clear. Genetic studies in the yeast Saccharomyces cerevisiae have shown that translation initiation factor eIF5-promoted hydrolysis of GTP bound to the 40 S ribosomal complex plays an essential role in AUG start codon selection by the 40 S subunit and that the two processes are tightly coupled. In order to ensure translation fidelity, hydrolysis of GTP bound to the 40 S preinitiation complex (40 · Met-tRNAi · eIF2 · GTP) must occur only when the complex has selected the AUG codon. However, since all the components required for GTP hydrolysis, namely eIF2, eIF5 and the 40 S ribosome, are present in the 40 S preinitiation complex, there must exist a mechanism by which eIF5-promoted GTP hydrolysis and, therefore, aberrant initiation is prevented prior to AUG selection by the ribosomal machinery. To understand the biochemical basis for this coupling, we have first characterized the requirements for the quantitative binding of initiator Met-tRNA to 40 S ribosomal subunits, in the absence of mRNA, to form a stable 40 S preinitiation complex. It was observed that three translation initiation factors, eIF1, eIF1A and eIF3 were together required to form a stable 40 S preinitiation complex and that all three factors remained bound to the 40 S complex. In such a 40 S preinitiation complex, eIF5-promoted GTP hydrolysis was inhibited by nearly 50%. However, when the 40 S preinitiation complex was positioned at the 5' capped-end of the mRNA in the presence of the initiation factor eIF4F, GTP hydrolysis was nearly completely prevented. In contrast, scanning of the mRNA by the 40 S preinitiation complex and positioning of the complex at the AUG codon (determined by toe-print analysis) resulted in restoration of eIF5-promoted GTP hydrolysis. Taken together, these results demonstrate the biochemical requirements for regulation of GTP hydrolysis and its coupling to the AUG selection process during translation initiation.