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

Abstract

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and by tryptophan-induced inhibition of Rho-mediated transcription termination. Previous studies indicated that tryptophan induction might involve leader peptide inhibition of ribosome release at the stop codon of tnaC, the coding region for the operon-specified leader peptide. In this study we examined tna operon expression in strains in which the structural gene for protein release factor 3, prfC, is either disrupted or overexpressed. We find that prfC inactivation leads to a two-to threefold increase in basal expression of the tna operon and a slight increase in induced expression. Overexpression of prfC has the opposite effect and reduces both basal and induced expression. These effects occur in the presence of glucose and cyclic AMP, and thus Rho-dependent termination rather than catabolite repression appears to be the event influenced by the prfC alterations. prfC inactivation also leads to an increase in basal tna operon expression in various rho and rpoB mutants but not in a particular rho mutant in which the basal level of expression is very high. The effect of prfC inactivation was examined in a variety of mutants with alterations in the tna leader region. Our results suggest that translation of tnaC is essential for the prfC effect. The tryptophan residue specified by tnaC codon 12, which is essential for induction, when replaced by another amino acid, allows the prfC effect. Introducing UAG or UAA stop codons rather than the normal tnaC UGA stop codon, in a strain with an inactive prfC gene, also leads to an increase in the basal level of expression. Addition of the drug bicyclomycin increases basal operon expression of all mutant strains except a strain with a tnaC-lacZ fusion. Expression in the latter strain is unaffected by prfC alterations. Our findings are consistent with the interpretation that ribosome release at the tnaC stop codon can influence tna operon expression.The tryptophanase (tnaAB) operon of Escherichia coli consists of two major structural genes, tnaA and tnaB, encoding tryptophanase and a tryptophan permease, respectively (7,27,29). Transcription of the tna operon is regulated by catabolite repression and by tryptophan-induced antitermination at Rhofactor-dependent termination sites in the leader region of the operon (Fig.

Rho-Dependent Transcription Termination in the tna Operon of Escherichia coli : Roles of the boxA Sequence and the rut Site

“…It was shown that a mutation that alters RF1 increased basal-level expression of the tna operon in strains with UAG or UAA as the tnaC stop codon, but not in strains with UGA as the stop codon (22). Additionally, inactivation of the structural gene for RF3 increased basal-level expression of the tna operon at least threefold (21,49), consistent with the role of RF3 in enhancing translation termination at UGA, UAG, and UAA stop codons (14,27). These results support the hypothesis that, in the presence of tryptophan, the nascent TnaC peptide inhibits ribosome release at the tnaC stop codon, thereby preventing Rho-dependent termination.…”

Amber (UAG) suppressors affected in UGA/UAA‐specific polypeptide release factor 2 of bacteria: genetic prediction of initial binding to ribosome preceding stop codon recognition

“…These stringent correlations between the suppressible codon and the species of altered RFs are thought to represent the selectivity of the stop codon by bacterial RF1 and RF2. Unlike RF1 and RF2, mutations in the RF3 gene (prfC) cause a suppression of all three stop codons Matsumura et al 1996;Yanofsky et al 1996;Crawford et al 1999). RF3 is thought to accelerate the dissociation of RF1 and RF2 from the ribosome in a GTP-dependent manner Pavlov et al 1997).…”

Ribosome Recycling Factor and Release Factor 3 Action Promotes TnaC-Peptidyl-tRNA Dropoff and Relieves Ribosome Stalling during Tryptophan Induction of tna Operon Expression in Escherichia coli

“…This result suggests that in the absence of RRF, a ribosome translating the tnaC Arg12 sequence is not liberated normally from the tnaC stop codon. It was also shown previously that the overproduction of RF3 influences the expression of tnaA of E. coli; however, the explanation for this observation was not known at that time (31). Previous studies also showed that RRF and RF3 were involved in peptidyl-tRNA dropoff from a translating ribosome (12,28).…”

“…RRF and EF-G as well as other factors, e.g., RF3 and IF3, then participate in the ribosome release/recycling process to provide ribosomal subunits for additional protein synthesis (13). Interestingly, the inactivation of prfC, the structural gene for RF3, has been reported to increase basal-level expression of the tna operon two-to threefold (31). Overexpression of prfC had the opposite effect: it reduced both the basal and induced levels of tna operon expression (31).…”

“…Interestingly, the inactivation of prfC, the structural gene for RF3, has been reported to increase basal-level expression of the tna operon two-to threefold (31). Overexpression of prfC had the opposite effect: it reduced both the basal and induced levels of tna operon expression (31). On the basis of this knowledge, experiments were performed to determine whether the overproduction of any of the translation factors, alone or in combination, would affect tna operon expression.…”