Studies on the promoter specificity determinants and modulators of the prokaryotic transcription machinery

Abstract

Bacteria adapt to a wide range of environmental conditions by synchronizing transcription with changes in the extracellular environment. This is achieved by the synchronized action of two-component systems, Extra Cytoplasmic Function (ECF) σ factors and myriad one-component systems that combine the roles of both sensors as well as transcription factors. While two-component systems correlate histidine kinase activity with changes in the transcription of target genes, in the case of ECF σ factors, this signal transduction is brought about by the release of free σ factors in the bacterial cell upon specific extracellular triggers. This, in turn, rapidly re-engineers the expression profile. One-component systems are simpler sensor/transcription modulators that operate on specific target proteins. Amongst these three mechanisms, the ECF σ factors, in particular, achieve both-specific expression of genes in a regulon as well as the magnitude of change in expression. This feature, alongside the finding that bacteria often have multiple ECF σ factors, suggests that the activity of ECF σ factors play a dominant role in governing the expression profile of a bacterial cell. The molecular determinants of both- specificity as well as transcription efficiency remain less understood despite substantial structural information on these systems that was reported over the past two years. Indeed, while the structures provided snapshots that could selectively rationalize specific biochemical observations, these could not rationalize the finding that non-conserved, unstructured segments in ECF σ factors played a significant functional role. The structural information on the transcription initiation complexes, however, provided a basis to design ‘rational’ biochemical experiments to evaluate the determinants governing ECF σ factor specificity for a given promoter element and the hypothesis that the linker between the two DNA-interacting domains in an ECF σ factor governs the efficiency of transcription initiation. These studies are reported in this thesis. Together, the work described in this thesis has two distinct applications. While specificity determinants in ECF σ factors provide a tool-box for synthetic biology applications, synthetic modulators, on the other hand, have the potential to form lead compounds for non-competitive inhibitors of RNAP activity. These studies thus provide a basis for more directed research from the perspective of engineering microbes with synthetic ECF σ factors. The feasibility of using chemical entities binding the RNAP interacting surface of a σ factor to modulate transcription could potentially lead to compounds that might find applications in therapeutic intervention.