PMID: 109437Jun 25, 1979

The purification and sequence of a temperature-sensitive tryptophan tRNA.

The Journal of Biological Chemistry
S P EisenbergM Yarus

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.

Related Concepts

DNA Sequence
Alkalescens-Dispar Group
RNase Pch 1
Mutation
RNA Conformation
Genomic Hybridization
Oligoribonucleotides
Alkaline Ribonuclease
Transfer RNA
Temperature

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