Rho-dependent transcription termination in the tna operon of Escherichia coli: roles of the boxA sequence and the rut site

Abstract

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and by tryptophan-induced transcription antitermination. Tryptophan induction prevents Rho-dependent transcription termination in the leader region of the operon. Induction requires translation of a 24-residue leader peptide-coding region, tnaC, containing a single, crucial Trp codon. Studies with a lacZ reporter construct lacking the tnaC-tnaA spacer region suggest that, in the presence of excess tryptophan, the TnaC leader peptide acts in cis on the ribosome translating tnaC to inhibit its release. The stalled ribosome is thought to block Rho's access to the transcript. In this paper we examine the roles of the boxA sequence and the rut site in Rho-dependent termination. Deleting six nucleotides (CGC CCT) of boxA or introducing specific point mutations in boxA results in high-level constitutive expression. Some constitutive changes introduced in boxA do not change the TnaC peptide sequence. We confirm that deletion of the rut site results in constitutive expression. We also demonstrate that, in each constitutive construct, replacement of the tnaC start codon by a UAG stop codon reduces expression significantly, suggesting that constitutive expression requires translation of the tnaC coding sequence. Addition of bicyclomycin, an inhibitor of Rho, to these UAG constructs increases expression, demonstrating that reduced expression is due to Rho action. Combining a boxA point mutation with rut site deletion results in constitutive expression comparable to that of a maximally induced operon. These results support the hypothesis that in the presence of tryptophan the ribosome translating tnaC blocks Rho's access to the boxA and rut sites, thereby preventing transcription termination.

FIG. 1

(A) Schematic diagram of the tnaA′-′lacZ fusion used in this study. This construct contains the tnaC leader region, the 220-nt spacer region, and the first 20 codons of tnaA joined to the ninth codon of lacZ. The mutagenized fragments in this study did not include the lacZ coding region. (B) Nucleotide sequence of boxA and the rut site (underlined). The TGA stop codon (boldface) is part of the boxA region. CAP, catabolite activator protein binding site.

FIG. 2

Predicted RNA secondary structure present at the end of the tnaC transcript. The RNA secondary structure was predicted using the MFOLD program of Zuker et al. (50, 51). The free energy of formation of the wild-type structure is −9.6 kcal/mol. Trp codon UGG and the stop codon UGA are in boldface. The UGA stop codon is part of the 9-nt sequence of boxA (CGC CCU UGA) in the tna operon of E. coli.

FIG. 3

Nucleotide and amino acid changes in tnaC and the TnaC peptide that do or do not affect tna operon expression. Nucleotide changes that do not lead to amino acid changes are underlined and are above the TnaC leader peptide sequence. The results come from this study, from references to , , and , and from unpublished data of M. Eshoo and C. Yanofsky.