Functional expression of a yeast ochre suppressor tRNA gene in Escherichia coli

Abstract

The yeast tRNA Tyr ochre suppressor SUP6 gene and a derivative of this gene in which the 14-bp intervening sequence has been deleted, SUP6Δ, have been examined for functional expression in Escherichia coli. The SUP6Δ, but not the SUP6, gene codes for a functional transfer RNA which has been shown to suppress both ochre and amber nonsense mutations in E. coli. Although the SUP6Δ fragment is contained within a 750-bp restriction fragment, isolated from Saccharomyces cerevisiae, and contains no encoded CCA, the primary transcript, which originates from an E.coli promoter, approx. 1000 bp upstream, is processed to a mature, functional transfer RNA. The pattern of suppression, i.e., suppression of both ochre and amber mutants, is characteristic of E. coli ochre suppressing tRNAs and is in contrast to the pattern observed in yeast, where only ochre mutations are suppressed. The SUP6Δ encoded tRNA, although coding for tRNA Tyr in yeast, is not charged solely with tyrosine in E. coli. The functional expression of this mutant eukaryotic transfer RNA gene in E. coli affords a unique opportunity for studies of expression of a gene coding for a stable RNA, in both a prokaryotic and an eukaryotic host.