Separation of Saccharomyces cerevisiae tRNAs on Two‐Dimensional Polyacrylamide Gels as Applied to Investigations on the Mutational Alterations of tRNA that Produce Nonsense Suppressors

Abstract

Nonsense suppressors of the yeast Saccharomyces cerevisiae have been found that cause the translational suppression of UAA (ochre) or UAG (amber) nonsense mutants of iso‐1‐cytochrome c by inserting tyrosine, serine or leucine at the site of the nonsense codon. This report describes the fractionation of tyrosine, serine and leucine tRNAs from yeast on two‐dimensional polyacrylamide gels, as well as a partial characterisation of these species. There is only one tyrosine tRNA in yeast, whilst the serine and leucine tRNAs identified in this study can between them decode all the serine and leucine codons which differ in only one base from either UAA or UAG. We did not characterise those serine and leucine tRNAs whose cognate codons differ in more than one base from the amber or ochre codon.Separation of the tRNA of individual yeast strains on two‐dimensional gels can be used in conjunction with RNA fingerprinting techniques to investigate whether the amber or ochre suppressor mutations of yeast determine structural changes in the tyrosine, serine or leucine tRNAs. This approach has already allowed an elucidation of the tRNATyr structural change determined by tyrosine‐inserting amber suppressor mutations at the SUP5 locus. Although it has, to date, only been successfully applied to the sequencing of yeast amber suppressors, it has also a potential application in structural studies of ochre suppressors. One of the leucine tRNAs analysed in this study contains an unidentified modified nucleotide in its anticodon, a minor component of interest as it may also be present in the anticodon of yeast ochre suppressor tRNAs and cause the restrictive recognition of UAA but not UAG codons. The possible anticodon structures of yeast ochre‐suppressing tRNAs are discussed in the light of present knowledge of codon‐anticodon pairing.