Functional analysis of spirochaetal promoters using a reconstituted transcription system

Abstract

Transcription is the first and most regulated step in gene expression. Transcription is catalysed by RNA polymerase (RNAP), a complex multi-subunit enzyme. RNAP can read regulatory signals encoded in the genomic DNA and respond to the changes in the concentration of substrate NTPs. However, most of the regulatory inputs are delivered to RNAP via accessory protein factors and regulatory RNAs. The transcription systems of model organisms such as Escherichia coli and Bacillus subtilis have been studied in great detail. In contrast, transcription systems of spirochetes are poorly understood. To study spirochaetal transcription, we selected Spirochaeta africana as a model organism. We expressed and purified components, reconstituted the core transcription system of S. africana in vitro and measured the activities of selected promoters and transcription factors. We measured the in vitro activity of S. africana and E. coli RNAPs at nine selected spirochaetal promoters and a control E. coli promoter at pH 7.5, which is close to intracellular pH in E. coli. Our transcription templates encoded fluorogenic RNA aptamer called Broccoli downstream the studied promoters. We monitored the transcription output by following the fluorescence levels of the RNA aptamer bound to a fluorogen. The activity of spirochaetal RNAP was several folds lower than that of E. coli RNAP at most promoters. We then measured the transcription activities of spirochaetal RNAP at pH 9, which is close to the optimal growth pH for these bacteria. Increasing pH significantly increased the activity of S. africana RNAP at several promoters bringing the overall activity profile closer to that of E. coli RNAP. Prior studies in our laboratory indicate that activities of some S. africana promoters are modulated by transcription factor CarD. We reproduced previously observed effects of CarD at two promoters and discovered an additional CarD activatable promoter. Overall, the activity profile of spirochaetal RNAP matched the E. coli RNAP profile best when the former enzyme was assayed at pH 9 in the presence of CarD. As a part of the investigation, we also used primer extension technique to map the transcription start sites for a subset of promoters to confirm their identities. Overall, our studies on spirochetes aim to enhance the understanding of their physiological mechanisms. This information can be used in the development of treatments for diseases propagated by pathogenic spirochetes, such as borreliosis.