Identification of transfer RNA suppressors in Escherichia coli. IV. Amber suppressor Su+6 a double mutant of a new species of leucine tRNA.
An Escherichia coli DNA fragment containing an Su+6 amber suppressor gene (supP) was cloned into a lambda gt lambda Ch vector by the shotgun method, selecting a Su+6 transducing phage lambda pSu+6. Through prophage integration followed by induction occurring at the transducing region of the lambda pSu+6 in Su- E. coli, a counterpart transducing phage carrying the wild-type allele (Su degrees 6) was isolated (lambda pSu degrees 6). The fingerprint of a tRNA encoded by lambda pSu degrees 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 degrees 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 degrees 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 degrees 6 tRNA gene constitutes a solitary tRNA transcription unit. Southern blotting showed only one copy of Su degrees 6 tRNA gene per haploid genome of E. coli. Since this single gene can mutate to the Su+6 suppressor, the Su degrees 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 degrees 6 tRNA gene.
D010582:Bacteriophage lambda / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D009154:Mutation; D012329:RNA, Bacterial / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics*; D013489:Suppression, Genetic; D014161:Transduction, Genetic
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D012343:RNA, Transfer