Escherichia coli Tryptophan Synthase: Synthesis of Catalytically Competent a Subunit in a Cell-Free System Containing Preacylated tRNAs

A cell-free protein biosynthesizing system prepared from Escherichia coli CF300 was found to synthesize E. coli tryptophan synthase a 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 a protein [Nichols, B. P., & Yanofsky, C. (1983) Methods Enzymol. 101, 155–164]. The synthesized tryptophan synthase a subunit was found to be indistinguishable from authentic a subunit protein when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and to have the same specific activity for catalyzing the conversion of indole → L-tryptophan by tryptophan synthase β₂subunit, 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-tRNA^Phe to the protein biosynthesizing system stimulated the production of functional a protein; the analogous result was obtained when valine was replaced by E. coli valyl-tRNA^Val. The ability of a misacylated tRNA to participate in a protein synthesis in this system was established by the use of E. coli phenylalanyl-tRNA^Val in the absence of added valine. Protein biosynthesis proceeded normally and gave a product having the approximate molecular weight of tryptophan synthase a subunit; as expected, this polypeptide lacked catalytic activity.