The purification and sequence of a temperature-sensitive tryptophan tRNA.
Escherichia coli can be temperature-sensitive due to a lesion in the gene for tRNATrp (Yanofsky, C., and Soll, L. (1977) J. Mol. Biol. 113, 663-677). Purification of tRNATrp from this strain (temperature-sensitive tRNATrp) was achieved by one of two methods. Either a combination of benzoylated DEAE-cellulose column chromatography and two-dimensional polyacrylamide gel electrophoresis, or hybridization to plasmid DNA covalently bound to cellulose (this is a recombinant plasmid carrying the gene for tRNATrp) and electrophoresis of the eluted material on a 10% polyacrylamide gel, produced isotopically pure tRNA. The sequence of the temperature-sensitive tRNATrp was determined by standard methods. We find that the sequence differs from that of wild type tRNATrp by a single residue; G in position 7 (G7) in wild type tRNATrp is A7 in temperature-sensitive tRNATrp. This base difference results in one less base pair in the CCA stem of the temperature-sensitive species. The effect of this base change in the in vitro and in vivo properties of tRNATrp (presented elsewhere (S.P. Eisenberg and M. Yarus, manuscript in preparation.)) are discussed.
D001483:Base Sequence; D004926:Escherichia coli / Q000032:analysis*; D009154:Mutation; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009843:Oligoribonucleotides / Q000032:analysis; D012343:RNA, Transfer / Q000302:isolation & purification; D006163:Ribonuclease T1; D012260:Ribonucleases; D013696:Temperature; D014364:Tryptophan
D009843:Oligoribonucleotides; D014364:Tryptophan; D012343:RNA, Transfer; D012260:Ribonucleases; D006163:Ribonuclease T1