The mechanism of tryptophan induction of tryptophanase operon expression: Tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNAPro

Feng Gong, Koichi Ito, Yoshikazu Nakamura, Charles Yanofsky

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Abstract

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. In a previous study, we reproduced the regulatory features of this operon observed in vivo by using an in vitro S-30 system. We also found that, under inducing conditions, the leader peptidyl-tRNA (TnaC-peptidyl-tRNAPro) is not cleaved; it accumulates in the S-30 reaction mixture. In this paper, we examine the requirements for TnaC-peptidyl-tRNAPro accumulation and cleavage, in vitro. We show that this peptidyl-tRNA remains bound to the translating ribosome. Removal of free tryptophan and addition of release factor 1 or 2 leads to hydrolysis of TnaC-peptidyl-tRNAPro and release of TnaC from the ribosome-mRNA complex. Release factor-mediated cleavage is prevented by the addition of tryptophan. TnaC of the ribosome-bound TnaC-peptidyl-tRNAPro was transferable to puromycin. This transfer was also blocked by tryptophan. Tests with various tryptophan analogs as substitutes for tryptophan revealed the existence of strict structural requirements for tryptophan action. Our findings demonstrate that the addition of tryptophan to ribosomes bearing nascent TnaC-peptidyl-tRNAPro inhibits both TnaC peptidyl-tRNAPro hydrolysis and TnaC peptidyl transfer. The associated translating ribosome therefore remains attached to the leader transcript where it blocks Rho factor binding and subsequent transcription termination.

Original languageEnglish (US)
Pages (from-to)8997-9001
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume98
Issue number16
DOIs
StatePublished - Jul 31 2001

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