Genetic interactions between a null allele of the RIT1 gene encoding an initiator tRNA-specific modification enzyme and genes encoding translation factors in Saccharomyces cerevisiae

Abstract

The Saccharomyces cerevisiae gene RIT1 encodes a phospho-ribosyl transferase that exclusively modifies the initiator tRNA (tRNA^Met _i) by the addition of a 2′-O-ribosyl phosphate group to Adenosine 64. As a result, tRNA^Met _i is prevented from participating in the elongation steps of protein synthesis. We previously showed that the modification is not essential for the function of tRNA^Met _i in the initiation of translation, since rit1 null strains are viable and show no obvious growth defects. Here, we demonstrate that yeast strains in which a rit1 null allele is combined with mutations in any of the genes for the three subunits of eukaryotic initiation factor-2 (eIF-2), or with disruption alleles of two of the four initiator methionine tRNA (IMT) genes, show synergistic growth defects. A multicopy plasmid carrying an IMT gene can alleviate these defects. On the other hand, introduction of a high-copy-number plasmid carrying the TEF2 gene, which encodes the eukaryotic elongation factor 1α (eEF-1α), into rit1 null strains with two intact IMT genes had the opposite effect, indicating that increased levels of eEF-1α are deleterious to these strains, presumably due to sequestration of the unmodified met-tRNA^Met _i for elongation. Thus, under conditions in which the components of the ternary met-tRNA^Met _i:GTP:eIF-2 complex become limiting or are functionally impaired, the presence of the 2′-O-ribosyl phosphate modification in tRNA^Met _i is important for the provision of adequate amounts of tRNA^Met _i for formation of this ternary complex.