Reproducing tna operon regulation in Vitro in an S-30 system: Tryptophan induction inhibits cleavage of TnaC peptidyl-tRNA

Feng Gong, C. Yanofsky

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

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'-'rpE 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.

Original languageEnglish
Pages (from-to)1974-1983
Number of pages10
JournalJournal of Biological Chemistry
Volume276
Issue number3
StatePublished - Jan 19 2001
Externally publishedYes

Fingerprint

Tryptophanase
Operon
Tryptophan
Transcription
Terminator Codon
Rho Factor
Catabolite Repression
Cell-Free System
Protein Sorting Signals
Codon
RNA, Transfer, Pro
Fusion reactions
Initiator Codon
peptidyl-tRNA
In Vitro Techniques
Ribosomes
Cyclic AMP
Escherichia coli
Messenger RNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Reproducing tna operon regulation in Vitro in an S-30 system : Tryptophan induction inhibits cleavage of TnaC peptidyl-tRNA. / Gong, Feng; Yanofsky, C.

In: Journal of Biological Chemistry, Vol. 276, No. 3, 19.01.2001, p. 1974-1983.

Research output: Contribution to journalArticle

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