Mutations affecting transcription pausing in the Bacillus subtilis pyr operon

Abstract

Pausing during transcription of the Bacillus subtilis pyr operon has been proposed to play a role in its regulation by attenuation. Substitution mutations in the B. subtilis pyr DNA specifying the 3'-terminal nucleotides of previously identified transcription pause sites substantially reduced pausing at these sites in vitro. This result confirms the general utility of this mutagenic strategy for studying transcriptional pausing. Pyrimidine-mediated repression in vivo of pyr-lacZ fusions containing some of these substitution mutations was substantially lower than observed with the wild-type pyr-lacZ fusions. However, these defects in regulation were correlated with alterations in the stability of the terminator stem-loop specified by the attenuator, rather than with their effects on transcriptional pausing in vitro.

RNA Polymerase Pausing Regulates Translation Initiation by Providing Additional Time for TRAP-RNA Interaction

“…The E. coli and Serratia marcescens trp leader pause RNAs that were identified in vivo were identical to those found in vitro (Landick et al, 1987); however, mutations that influence pausing in vitro and gene expression in vivo have not been reported. Mutations leading to in vitro RNAP pausing defects did not appear to influence gene expression of the B. subtilis glyQS (Grundy and Henkin, 2004) or pyr (Zhang et al, 2005) operons in vivo; however, in both cases, new RNAP pause sites arose. In the case of the pyr operon, it is possible that the mutations in the nascent transcript that altered pausing in vitro led to changes in the antiterminator structure, potentially overshadowing the effect of the pause defect in vivo (Zhang et al, 2005).…”

“…Mutations leading to in vitro RNAP pausing defects did not appear to influence gene expression of the B. subtilis glyQS (Grundy and Henkin, 2004) or pyr (Zhang et al, 2005) operons in vivo; however, in both cases, new RNAP pause sites arose. In the case of the pyr operon, it is possible that the mutations in the nascent transcript that altered pausing in vitro led to changes in the antiterminator structure, potentially overshadowing the effect of the pause defect in vivo (Zhang et al, 2005).…”

“…A central feature of several transcription attenuation mechanisms is that formation of an antiterminator structure prevents formation of a mutually exclusive intrinsic terminator hairpin, thereby promoting transcription readthrough into the structural genes. Regulatory molecules that interact with the nascent transcript, such as translating ribosomes (Winkler and Yanofsky, 1981), RNA binding proteins (Yakhnin and Babitzke, 2002;Zhang et al, 2005), tRNA (Grundy and Henkin, 2004), or metabolites (Mironov et al, 2002;Winkler et al, 2002), have been shown to be responsible for the decision as to which of the mutually exclusive structures form. Because the regulatory molecule must bind to the nascent transcript before the terminator structure forms, a relatively short window of opportunity exists for this interaction to take place.…”

“…Three general classes of RNAP pause sites have been described: promoter proximal, hairpin dependent, and hairpin independent (Kainz and Roberts, 1995;Artsimovitch and Landick, 2000a;Landick, 2004). RNAP pause sites have been identified in the untranslated leaders of the E. coli trp (Winkler and Yanofsky, 1981), his (Mooney et al, 1998), tna (Gong and Yanofsky, 2003), and S10 (Sha et al, 1995) operons, the bacteriophage lambda late transcript (Marr and Roberts, 2000), and the B. subtilis pyr (Zhang et al, 2005), glyQS (Grundy and Henkin, 2004), and trp (Yakhnin and Babitzke, 2002) operons. RNAP pausing has been documented for several eukaryotic genes as well; promoter-proximal pausing occurs for several Drosophila heat-shock genes (Rasmussen and Lis, 1993), whereas hairpin-dependent pausing occurs during transcription of HIV (Palangat et al, 1998).…”

Regulation of Pyrimidine Biosynthetic Gene Expression in Bacteria: Repression without Repressors

“…These findings demonstrated that transcription pausing could play an important role in the regulation of the pyr operon, but they did not demonstrate that it actually does so in vivo. In a subsequent study, Zhang et al attempted to evaluate the function of transcription pausing in the pyr operon in vivo by constructing and analyzing mutations that greatly reduce pausing (177). The mutations changed selected pyrimidine nucleotides to purine nucleotides near pause sites, which was shown to substantially reduce transcription pausing at the mutant site in vitro.…”