Evidence suggesting cis action by the TnaC leader peptide in regulating transcription attenuation in the tryptophanase operon of Escherichia coli

Abstract

Expression of the tryptophanase (tna) operon in Escherichia coli is regulated by catabolite repression and transcription attenuation. Elevated levels of tryptophan induce transcription antitermination at one or more Rho factor-dependent termination sites in the leader region of the operon. Induction requires translation of a 24-residue coding region, tnaC, located in the 319-nucleotide transcribed leader region preceding tnaA, the structural gene for tryptophanase. In the present paper, we show that two bacterial species that lack tryptophanase activity, Enterobacter aerogenes and Salmonella typhimurium, allow tryptophanase induction and tna operon regulation when they carry a plasmid containing the E. coli tna operon. The role of tnaC in induction was examined by introducing mutations in a 24-nucleotide segment of tnaC of E. coli surrounding and including the crucial Trp codon 12. Some mutations resulted in a noninducible phenotype; these mostly introduced nonconservative amino acid substitutions in TnaC. Other mutations had little or no effect; these generally were in third positions of codons or introduced conservative amino acid replacements. A tryptophan-inserting, UGA-reading glutamine suppressor tRNA was observed to restore partial regulation when Trp codon 12 of tnaC was changed to UGA. Stop codons introduced downstream of Trp codon 12 in all three reading frames established that induction requires translation in the natural tnaC reading frame. Our findings suggest that the TnaC leader peptide acts in cis to prevent Rho-dependent termination.Tryptophanase is a catabolic enzyme produced by many bacterial species. This enzyme degrades tryptophan to indole, pyruvate, and ammonia and allows the organism to utilize tryptophan as a source of nitrogen or of carbon and energy (13,24). Tryptophanase also catalyzes the reverse reaction (43). In Escherichia coli, the tna operon consists of two major structural genes, tnaA, encoding tryptophanase (9), and tnaB, encoding a tryptophan permease (5,32,45). The leader region of the tna operon contains a region, designated tnaC, encoding a 24-amino-acid residue peptide. Transcription of the structural gene region of the tna operon is responsive to the presence of tryptophan but not other amino acids; addition of tryptophan to a growth medium results in a 10-to 100-fold elevation of the tryptophanase level (39). Although the precise mechanism of induction is unknown, previous studies established that addition of tryptophan induces transcription antitermination at one or more Rho factor-dependent termination sites located in the transcribed leader region of the operon (39, 40). Mutations in rho can elevate operon expression (39); also, deletion of a putative Rho utilization (rut) site, located immediately after the tnaC stop codon, resulted in constitutive operon expression (12). The major sites of termination were determined in vitro and in vivo and correspond to positions ϩ234 and ϩ291 relative to the start of transcription (38).Translation of tnaC appears to be required ...

Loss of overproduction of polypeptide release factor 3 influences expression of the tryptophanase operon of Escherichia coli

“…Inhibition of ribosome release might block Rho's access to the rut site on the tna transcript and thereby prevent transcription termination. The most compelling evidence supporting this hypothesis is the finding that amino acid substitutions in TnaC in the vicinity of the crucial Trp residue eliminate tryptophan-induced antitermination, whereas synonymous codon changes in the codons for these or other residues in the same segment of the polypeptide allow tryptophan-induced transcription antitermination (12). Alternative plausible explanations are that TnaC interacts directly with Rho to inhibit its action, that TnaC interferes with the ability of NusG or some other factor to activate Rho (5,6,23,32), or that TnaC prevents some event at the box A sequence that is required for Rho action (10,34).…”

“…Several lines of evidence suggest that the leader region is naturally programmed for transcription termination (11)(12)(13)(28)(29)(30). Thus, Rho-dependent termination in this region is the predominant event when cells lack inducing levels of tryptophan.…”

“…Our currently favored hypothesis for the mechanism of induction is that in the presence of excess tryptophan TnaC acts in cis on the ribosome translating tnaC to prevent Rho-dependent transcription termination (12). TnaC could act by inhibiting ribosome release at the tnaC stop codon (12). Inhibition of ribosome release might block Rho's access to the rut site on the tna transcript and thereby prevent transcription termination.…”

“…For induction to occur, synthesis of the TnaC leader peptide is required. Our currently favored hypothesis for the mechanism of induction is that in the presence of excess tryptophan TnaC acts in cis on the ribosome translating tnaC to prevent Rho-dependent transcription termination (12). TnaC could act by inhibiting ribosome release at the tnaC stop codon (12).…”

Pheromone cCF10 and plasmid pCF10‐encoded regulatory molecules act post‐transcriptionally to activate expression of downstream conjugation functions

“…In other systems, effects of cis-acting peptides on ribosome function have been well documented (Gish and Yanofsky, 1995). Inhibition of peptidyl transferase activity by peptides from the leader regions of the cat and erm antibiotic-resistance genes in B. subtilis has been demonstrated (Lovett, 1994).…”