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J. Biol. Chem., Vol. 276, Issue 3, 1974-1983, January 19, 2001
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From the Department of Biological Sciences, Stanford University,
Stanford, California 94305
Expression of the tryptophanase (tna)
operon of Escherichia coli is regulated by catabolite
repression and tryptophan-induced transcription antitermination.
Catabolite repression regulates transcription initiation, whereas
excess tryptophan induces antitermination at Rho
factor-dependent termination sites in the leader region of
the operon. Synthesis of the leader peptide, TnaC, is essential for
antitermination. BoxA and rut sites in the
immediate vicinity of the tnaC stop codon are required for
termination. In this paper we use an in vitro S-30
cell-free system to analyze the features of tna operon
regulation. We show that transcription initiation is cyclic AMP
(cAMP)-dependent and is not influenced by tryptophan. Continuation of transcription beyond the leader region requires the
presence of inducing levels of tryptophan and synthesis of the TnaC
leader peptide. Using a tnaA'-'trpE fusion, we demonstrate that induction results in a 15-20-fold increase in synthesis of the
tryptophan-free TnaA-TrpE fusion protein. Replacing Trp codon 12 of
tnaC by an Arg codon, or changing the tnaC
start codon to a stop codon, eliminates induction. Addition of
bicyclomycin, a specific inhibitor of Rho factor action, substantially
increases basal level expression. Analyses of tna mRNA
synthesis in vitro demonstrate that, in the absence of
inducer transcription is terminated and the terminated transcripts are
degraded. In the presence of inducer, antitermination increases the
synthesis of the read-through transcript. TnaC synthesis is observed in
the cell-free system. However, in the presence of tryptophan, a
peptidyl-tRNA also appears, TnaC-tRNAPro. Our findings
suggest that inducer acts by preventing cleavage of TnaC peptidyl-tRNA.
The ribosome associated with this newly synthesized peptidyl-tRNA
presumably stalls at the tnaC stop codon, blocking Rho's
access to the BoxA and rut sites, thereby
preventing termination. 1-Methyltryptophan also is an effective inducer
in vitro. This tryptophan analog is not incorporated into TnaC.
Reproducing tna Operon Regulation in
Vitro in an S-30 System
TRYPTOPHAN INDUCTION INHIBITS CLEAVAGE OF TnaC
PEPTIDYL-tRNA*
*
This work was supported by Grant GM09738 (to C. Y.)
from the United States Public Health Service.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 650-725-1835;
Fax: 650-725-8221; E-mail: yanofsky@cmgm.stanford.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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