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

Feng Gong,Charles Yanofsky

doi:10.1074/jbc.m008892200

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'-'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.

Highlights

The enzyme tryptophanase catalyzes the degradation of Ltryptophan to indole, pyruvate, and ammonia
Cyclic AMP-dependent Expression of a tnaCЈ-ЈlacZ Fusion in an S-30 System—In previous studies it was shown that initiation of transcription of the tna operon is regulated by catabolite repression [8]
Addition of tryptophan increased ␤-galactosidase production less than 2-fold. These results suggest that expression of tnaCЈ-ЈlacZ construct in this cell-free system is cyclic AMP (cAMP)-dependent, and that tryptophan produced by protein turnover provides most of the tryptophan required for ␤-galactosidase synthesis

Summary

Introduction

The enzyme tryptophanase catalyzes the degradation of Ltryptophan to indole, pyruvate, and ammonia. Basal and Induced Expression with the tnaAЈ-ЈtrpE Construct—To study tryptophan induction of tna operon expression in vitro, we prepared a special construct, pGF4, containing the intact tna promoter-leader region followed by a translational fusion of the initial segment of tnaA fused to trpE of E. coli (Fig. 1B).

Results

Conclusion