Escherichia coli tryptophan synthase: synthesis of catalytically competent .alpha. subunit in a cell-free system containing preacylated tRNAs

Abstract

A cell-free protein biosynthesizing system prepared from Escherichia coli CF300 was found to synthesize E. coli tryptophan synthase .alpha. subunit in a time-dependent manner when programmed with pBN69 plasmid DNA. This plasmid contains the trp promoter from Serratia marcescens adjacent to the coding region of E. coli tryptophan synthase .alpha. protein [Nicholas, B. P., and Yanofsky, C. (1983) Methods Enzymol. 101, 155-164]. The synthesized tryptophan synthase .alpha. subunit was found to be indistinguishable from authentic .alpha. subunit protein when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and to have the same specific activity for catalyzing the conversion of indole.fwdarw.L-tryptophan by tryptophan synthase .beta.2 subunit, as well as the conversion of indole+glyceraldehyde 3-phosphate to indole-3-glycerol phosphate. In the absence of exogenously added phenylalanine, admixture of E. coli phenylalanyl-tRNAPhe to the protein biosynthesizing system stimulated the production of functional .alpha. protein; the analogous result was obtained when valine was replaced by E. coli valyl-tRNAVal. The ability of a misacylated tRNA to participate in .alpha. protein synthesis in this system was established by the use of E. coli phenylalanyl-tRNAVal in the absence of added valine. Protein biosynthesis proceeded normally and gave a product having the approximate molecular weight of tryptophan synthase .alpha. subunit; as expected, this polypeptide lacked catalytic activity.