Identification of transfer RNA suppressors in Escherichia coli. IV. Amber suppressor su+6 a double mutant of a new species of leucine tRNA

Abstract

An Escherichia coli DNA fragment containing an Su+6 amber suppressor gene (supP) was cloned into a λgtλCh vector by the shotgun method, selecting a Su+6 transducing phage λpSu+6. Through prophage integration followed by induction occurring at the transducing region of the λpSu+6 in Su- E. coli, a counterpart transducing phage carrying the wild-type allele (Su{ring operator}6) was isolated (λpSu{ring operator}6). The fingeprint of a tRNA encoded by λpSu{ring operator}6 was identical to that of an unidentified tRNAE previously reported (Ikemura & Ozeki, 1977). The cloverleaf structure of this tRNA was determined by combining the results of tRNA analysis and DNA sequencing of the gene. Judging from the anticodon of 5′-CAA-3′, Su{ring operator}6 tRNA was identified as a new type of leucine isoacceptor in E. coli. Unlike other suppressors analyzed, Su+6 tRNA differed by two nucleotides from Su{ring operator}6 tRNA; one at the anticodon (CAA to CUA) and the other at the junction of D- and anticodon-stem (A27 to G27). DNA sequence analysis revealed that a single stretch of tRNA is flanked by the putative sequences of promoter and terminator. Thus a single copy of the Su{ring operator}6 tRNA gene constitutes a solitary tRNA transcription unit. Southern blotting showed only one copy of Su{ring operator}6 tRNA gene per haploid genome of E. coli. Since this single gene can mutate to the Su+6 suppressor, the Su{ring operator}6 leucine tRNA may be accounted as a dispensable species among the leucine isoacceptor tRNAs. Two possible open reading frames are found immediately following the Su{ring operator}6 tRNA gene. © 1984.