Global 6S transcriptional regulation responds to environmental conditions by metabolite cofactor sensing

Resource type
Thesis type
(Thesis) M.Sc.
Date created
The E. coli 6S RNA competes with σ70-dependent housekeeping DNA promoters for binding to RNA polymerase holoenzyme (RNAP:σ70). When bound to RNAP:σ70, the 6S is used as a template to synthesize a short product RNA (pRNA; usually, 13 nt long). This pRNA changes the 6S structure and triggers the release of the 6S:pRNA from RNAP, allowing DNA-dependent transcription to resume. 6S turnover in high nutrient conditions is extremely rapid, whereas 6S turnover in low nutrient conditions is very slow. This leads to a large accumulation of inhibited RNAP:σ70 complex during stationary phase. As pRNA initiates synthesis with ATP, it is likely that the 6S release rate might be strongly dependent on ATP levels as a proxy for sensing cellular metabolic state. By purifying 6S:pRNA complexes using an RNA Mango aptamer tag and by generating pRNA-cDNA hybrids, I first demonstrate that 6S 5' maturation during exponential growth is simultaneous with 6S:pRNA formation, and that longer 6S products are bound and released. More importantly, I then find a dramatic accumulation of capped pRNA products during stationary phase. This indicates that ATP levels are low enough for non-canonical initiator nucleotides (NCINs) such as NAD+ and NADH to serve as pRNA initiators. In vitro, I also found that mutating the 6S template sequence immediately upstream of the transcriptional start site can result in either increased or decreased levels of NCIN capping, which suggests that evolution has refined the biological 6S sequence for an optimal capping rate in low nutrient conditions. Robust NCIN-initiated pRNA synthesis may thus be essential for cell viability when ATP concentrations are extremely low.
89 pages.
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Supervisor or Senior Supervisor
Thesis advisor: Unrau, Peter
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