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The kinetics of formation of complexes between Escherichia coli RNA polymerase and the rrnB P1 and P2 promoters of Bacillus subtilis

University of British Columbia

In bacteria, a change in the growth rate (≥ 0.7 doublings per hour) is accompanied by a directly proportional change in the steady-state intracellular concentration of ribosomes. This growth rate dependent control of ribosome biosynthesis is apparently regulated at the level of ribosomal RNA (rRNA) transcription initiation by a feedback inhibition mechanism. One proposed effector of this feedback inhibition has been guanosine tetraphosphate (ppGpp). The effects of ppGpp on the kinetics of formation and dissociation of heparin resistant complexes between Escherichia coli RNA polymerase and the rrnB P1 (non growth rate regulated) and P2 (growth rate regulated) promoters from Bacillus subtilis were investigated as a function of temperature using a gel retardation assay. The results from this thesis suggest that the formation of polymerase/promoter complexes proceeded by way of three kinetically significant reaction steps. The initial bimolecular collision between free RNA polymerase and the promoter led to the formation of a heparin sensitive (HS) complex, which subsequently isomerized to an intermediate (HR1) and then final (HR2) heparin resistant complex. The temperature dependences of the forward isomerization rate constant (kf), and the (overall) second-order association rate constant (ka), were inconsistent with the proposal that free RNA polymerase and promoter fragments existed in rapid equilibrium with HS. If this rapid equilibrium existed, then the predicted equilibrium constant for binding (K1 = ka/kf) would be exothermic, contradicting the temperature dependence of KI observed at other promoters. Consequently, it was proposed that a sequential mode of binding best described the bimolecular collision. The gel retardation assay described in this thesis examined the effects of ppGpp at a select number of steps in the overall path of transcription initiation. For the promoters in question, this analysis provided direct information regarding the formation of the HS and HR1 complexes, and indirect information regarding the formation of HR2 and initiated ternary complexes. The cumulative results from studies of the B. subtilis and E. coli rrnB PI and P2 promoters would suggest that ppGpp does not act to differentially inhibit transcription initiation at any of the steps investigated.

Text

2011-03-12

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http://hdl.handle.net/2429/32383

Microbiology and Immunology

University of British Columbia

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