Escherichia coli tryptophan synthase: Synthesis of catalytically competent α subunit in a cell-free system containing preacylated tRNAs

Robert C. Payne, Sidney Hecht, Sidney M. Hecht

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

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 β2 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 a protein; the analogous result was obtained when valine was replaced by E. coli valyl-tRNAVal. The ability of a misacylated tRNA to participate in a protein synthesis in this system was established by the use of E. coli phehylalanyl-tRNAVal 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.

Original languageEnglish (US)
Pages (from-to)3197-3205
Number of pages9
JournalBiochemistry
Volume26
Issue number11
StatePublished - 1987
Externally publishedYes

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Tryptophan Synthase
Cell-Free System
Transfer RNA
Escherichia coli
RNA, Transfer, Val
Valine
Proteins
Plasmids
Glyceraldehyde 3-Phosphate
RNA, Transfer, Phe
Serratia marcescens
Biosynthesis
Protein Subunits
Protein Biosynthesis
Electrophoresis
Phenylalanine
Tryptophan
Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis
Catalyst activity

ASJC Scopus subject areas

  • Biochemistry

Cite this

Escherichia coli tryptophan synthase : Synthesis of catalytically competent α subunit in a cell-free system containing preacylated tRNAs. / Payne, Robert C.; Hecht, Sidney; Hecht, Sidney M.

In: Biochemistry, Vol. 26, No. 11, 1987, p. 3197-3205.

Research output: Contribution to journalArticle

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