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1 Department of Biology, Indiana University, Bloomington,
Indiana 47405 USA;
2 Molecular and Cell Biology Program, The
University of Texas at Dallas, Richardson, Texas 75083 USA
We have isolated and characterized two suppressor genes,
SUI4 and SUI5, that can initiate translation in the
absence of an AUG start codon at the HIS4 locus in
Saccharomyces cerevisiae. Both suppressor genes are dominant in
diploid cells and lethal in haploid cells. The SUI4 suppressor
gene is identical to the GCD11 gene, which encodes the 
subunit of the eIF-2 complex and contains a mutation in the
G2 motif, one of the four signature motifs that characterizes
this subunit to be a G-protein. The SUI5 suppressor gene is
identical to the TIF5 gene that encodes eIF-5, a translation
initiation factor known to stimulate the hydrolysis of GTP bound to
eIF-2 as part of the 43S preinitiation complex. Purified mutant eIF-5
is more active in stimulating GTP hydrolysis in vitro than wild-type
eIF-5, suggesting that an alteration of the hydrolysis rate of GTP
bound to the 43S preinitiation complex during ribosomal scanning allows
translation initiation at a non-AUG codon. Purified mutant eIF-2
complex is defective in ternary complex formation and this defect
correlates with a higher rate of dissociation from charged
initiator-tRNA in the absence of GTP hydrolysis. Biochemical
characterization of SUI3 suppressor alleles that encode mutant
forms of the 
subunit of eIF-2 revealed that these mutant eIF-2
complexes have a higher intrinsic rate of GTP hydrolysis, which is
eIF-5 independent. All of these biochemical defects result in
initiation at a UUG codon at the his4 gene in yeast. These
studies in light of other analyses indicate that GTP hydrolysis that
leads to dissociation of eIF-2 · GDP from the initiator-tRNA in
the 43S preinitiation complex serves as a checkpoint for a 3-bp
codon/anticodon interaction between the AUG start codon
and the initiator-tRNA during the ribosomal scanning process.
[Key Words: GTP hydrolysis; translation initiation; ribosomal scanning; AUG selection; eIF-2; eIF-5]
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