The purification and sequence of a temperature-sensitive tryptophan tRNA

Abstract

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.