Analysis of tryptophanase operon expression in vitro: accumulation of TnaC-peptidyl-tRNA in a release factor 2-depleted S-30 extract prevents Rho factor action, simulating induction.
Expression of the tryptophanase (tna) operon in Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. The key feature of this antitermination mechanism has been shown to be the retention of uncleaved TnaC-peptidyl-tRNA in the translating ribosome. This ribosome remains stalled at the tna stop codon and blocks the access of Rho factor to the tna transcript, thereby preventing transcription termination. In normal S-30 preparations, synthesis of a TnaC peptide containing arginine instead of tryptophan at position 12 (Arg(12)-TnaC) was shown to be insensitive to added tryptophan, i.e. Arg(12)-TnaC-peptidyl-tRNA was cleaved, and there was normal Rho-dependent transcription termination. When the S-30 extract used was depleted of release factor 2, Arg(12)-TnaC-tRNA(Pro) was accumulated in the absence or presence of added tryptophan. Under these conditions the accumulation of Arg(12)-TnaC-tRNA(Pro) prevented Rho-dependent transcription termination, mimicking normal induction. Using a minimal in vitro transcription system consisting of a tna template, RNA polymerase, and Rho, it was shown that RNA sequences immediately adjacent to the tnaC stop codon, the presumed boxA and rut sites, contributed most significantly to Rho-dependent termination. The tna boxA-like sequence appeared to serve as a segment of the Rho "entry" site, despite its likeness to the boxA element.
Gong
Feng
F
Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA.
Yanofsky
Charles
C
D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003062:Codon; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009876:Operon; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012234:Rho Factor / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic; D014364:Tryptophan / Q000737:chemistry; D014368:Tryptophanase / Q000096:biosynthesis* / Q000235:genetics*
D001426:Bacterial Proteins; D003062:Codon; D029968:Escherichia coli Proteins; D012234:Rho Factor; C081473:tnaC protein, E coli; D012313:RNA; D014364:Tryptophan; D001120:Arginine; D012321:DNA-Directed RNA Polymerases; D014368:Tryptophanase