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PMID: 11470925 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

The mechanism of tryptophan induction of tryptophanase operon expression: tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNA(Pro).

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 98 ·No. 16 ·2001-07-31 ·Pages 8997-9001

Gong F, Ito K, Nakamura Y, Yanofsky C

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-tRNA(Pro)) is not cleaved; it accumulates in the S-30 reaction mixture. In this paper, we examine the requirements for TnaC-peptidyl-tRNA(Pro) 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-tRNA(Pro) 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-tRNA(Pro) 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-tRNA(Pro) inhibits both TnaC peptidyl-tRNA(Pro) 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.

MeSH Terms
Bacterial Proteins/genetics,metabolism Escherichia coli Proteins Hydrolysis Operon Puromycin/antagonists & inhibitors RNA, Transfer, Amino Acyl/metabolism RNA, Transfer, Pro/metabolism Tryptophan/physiology Tryptophanase/genetics
Chemicals
Bacterial Proteins Escherichia coli Proteins RNA, Transfer, Amino Acyl RNA, Transfer, Pro tRNA, peptidyl- tnaC protein, E coli Puromycin Tryptophan Tryptophanase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Gong F
Department of Biological Sciences, Stanford University, Stanford, CA 94305; and Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedal, Minato-Ku, Tokyo 108-8639, Japan.
Ito K
Nakamura Y
Yanofsky C
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.171299298
Published
2001-07-31
Epub
2001-00-24
Pages
8997-9001
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC55362
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.171299298 PMC Full Text View journal details
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