Escherichia coli tryptophan synthase: synthesis of catalytically competent alpha 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 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 [Nichols, B.P., & 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----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.
Payne
R C
RC
Nichols
B P
BP
Hecht
S M
SM
D002474:Cell-Free System; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D010957:Plasmids; D014176:Protein Biosynthesis / Q000187:drug effects; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D013095:Spermidine / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014367:Tryptophan Synthase / Q000096:biosynthesis / Q000235:genetics*
D046911:Macromolecular Substances; D012346:RNA, Transfer, Amino Acyl; D012321:DNA-Directed RNA Polymerases; D014367:Tryptophan Synthase; D008274:Magnesium; D013095:Spermidine