THE YEAST INITIATOR TRANSFER RNA(MET) CAN ACT AS AN ELONGATOR TRANSFER RNA(MET) IN-VIVO

Saccharomyces cerevisiae uses two different methionine accepting tRNAs during protein synthesis. One, tRNAi Met, is used exclusively during the initiation of translation whereas the other, tRNAm Met, is used during the elongation of translation. To study the unique features of each methionine tRNA species, we constructed yeast strains with null alleles of the five elongator methionine tRNA (EMT) genes and strains with null alleles of the four initiator methionine tRNA (IMT) genes, respectively. Consequently, growth of these strains wasdependent either on a tRNAm Met or a tRNAi Met, respectively, encoded from a plasmid-derived gene. For both null mutants, the plasmid carrying the wild-type gene can be selected against and exchanged for another plasmid derived EMT or IMT gene (wild-type or mutant). A high gene dosage of the wild-type IMT gene could restore growth to the elongator-depleted strain. However, wild-type EMT genes in a high gene dosage never restored growth of the initiator depleted strain. Thus, the elongator tRNAMet is much more restricted to participate in the initiation of translation than the initiator tRNAMet is restricted to participate in the elongation process. Using the twonull mutants, we have identified tRNAm Met mutants, which show reduced elongator activity, and tRNAi Met mutants, with improved elongator activity in the elongator depleted strain. Also, tRNAm Met mutants that function as an initiator tRNA in the initiator depleted strain were identified. From this mutant analysis, we showed that the conserved U/rT at position 54 of the elongator tRNAMet is an important determinant for an elongator tRNA. The most important determinant for an initiator was shown to be the acceptor stem and especially the conserved A1 · U72 base-pair. Mutant tRNAs, with reduced activity in either process, were investigated for enhanced activity during overproduction of the α and β-subunits of the eukaryotic initiation factor 2 (eIF-2) or the eukaryotic elongation factor 1α (eEF-1α). The data suggest that the U/rT of the elongator at position 54 is important for eEF-1α recognition and that the acceptor stem of the initiator is important for eIF-2 recognition. © 1993 Academic Press Limited.