i6A-deficient tRNA from an Antisuppressor Mutant of Saccharomyces cerevisiae

Abstract

The determination of the functional roles of modified nucleotides in tRNA has been the goal of numerous investigations (most recently reviewed by McCloskey and Nishimura 1977; Agris and Söll 1977). Nearly all attempts to assess minor base function have suffered from the same shortcomings. First, mutations in tRNA modifying enzymes have been difficult to select. Second, the use of in vitro techniques to create specific alterations in tRNA modification and assess their effects on tRNA function may lead to ambiguous results (see Discussion).

Our goal was to screen for strains of the yeast Saccharomyces cerevisiae with mutations in genes for tRNA modifying enzymes. The genetic screening for these mutants was based on the probable correlation between loss of base modification and reduction in the efficiency of suppression of a UAA nonsense suppressing tRNA. The existence of such a correlation was suggested by early in vitro findings (Gefter and Russell 1969) and has since been supported by recent in vivo studies (Colby et al. 1976; Marinus et al. 1975).

We have isolated a mutant that contains 1.5% of the normal tRNA complement of i⁶A (isopentenyladenosine). This mutant affords the opportunity to compare in vitro results with in vivo observations. The mutation, which has been designated mod5-1, reduces the suppression by a dominant UAA suppressor, SUP7-1 (Gilmore 1967), so that only the more easily suppressed of several UAA mutations are suppressed. SUP7-1 is one of several efficient tyrosine-inserting UAA suppressors and most probably codes for an altered tRNA^Tyr (Gilmore et al. 1971;...