HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 3, 1974-1983, January 19, 2001

This Article Full Text Full Text (PDF) All Versions of this Article: 276/3/1974    most recent M008892200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gong, F. Articles by Yanofsky, C. Search for Related Content PubMed PubMed Citation Articles by Gong, F. Articles by Yanofsky, C.

Reproducing tna Operon Regulation in Vitro in an S-30 System
TRYPTOPHAN INDUCTION INHIBITS CLEAVAGE OF TnaC PEPTIDYL-tRNA^*

Feng Gong and Charles Yanofsky

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-tRNA^Pro. 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.

This article has been cited by other articles:

				M. Beringer Modulating the activity of the peptidyl transferase center of the ribosome RNA, May 1, 2008; 14(5): 795 - 801. [Abstract] [Full Text] [PDF]

				L. R. Cruz-Vera, A. New, C. Squires, and C. Yanofsky Ribosomal Features Essential for tna Operon Induction: Tryptophan Binding at the Peptidyl Transferase Center J. Bacteriol., April 15, 2007; 189(8): 3140 - 3146. [Abstract] [Full Text] [PDF]

				M. Gong, F. Gong, and C. Yanofsky Overexpression of tnaC of Escherichia coli Inhibits Growth by Depleting tRNA2Pro Availability. J. Bacteriol., March 1, 2006; 188(5): 1892 - 1898. [Abstract] [Full Text] [PDF]

				F. Gong and C. Yanofsky A Transcriptional Pause Synchronizes Translation with Transcription in the Tryptophanase Operon Leader Region J. Bacteriol., November 1, 2003; 185(21): 6472 - 6476. [Abstract] [Full Text] [PDF]

				D. M. Janzen, L. Frolova, and A. P. Geballe Inhibition of Translation Termination Mediated by an Interaction of Eukaryotic Release Factor 1 with a Nascent Peptidyl-tRNA Mol. Cell. Biol., December 15, 2002; 22(24): 8562 - 8570. [Abstract] [Full Text] [PDF]

				F. Gong and C. Yanofsky Instruction of Translating Ribosome by Nascent Peptide Science, September 13, 2002; 297(5588): 1864 - 1867. [Abstract] [Full Text] [PDF]

				F. Gong and C. Yanofsky 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 J. Biol. Chem., May 3, 2002; 277(19): 17095 - 17100. [Abstract] [Full Text] [PDF]

				M.-K. Oh, L. Rohlin, K. C. Kao, and J. C. Liao Global Expression Profiling of Acetate-grown Escherichia coli J. Biol. Chem., April 5, 2002; 277(15): 13175 - 13183. [Abstract] [Full Text] [PDF]

				F. Gong, K. Ito, Y. Nakamura, and C. Yanofsky The mechanism of tryptophan induction of tryptophanase operon expression: Tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNAPro PNAS, July 19, 2001; (2001) 171299298. [Abstract] [Full Text] [PDF]

				F. Gong, K. Ito, Y. Nakamura, and C. Yanofsky The mechanism of tryptophan induction of tryptophanase operon expression: Tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNAPro PNAS, July 31, 2001; 98(16): 8997 - 9001. [Abstract] [Full Text] [PDF]