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A kinetic analysis of transcription initiation by the Bacillus subtilis sigma-43 RNA polymerase : the effect of the delta subunit

University of British Columbia

The initiation of transcription by the Bacillus subtilis sigma-M3 RNA polymerase at two Bacillus phage ɸ29 promoters and the effect of the delta subunit on initiation have been investigated by an in vitro kinetic analysis. The templates for the analysis were plasmids which carried the ɸ29 A2 or G2 promoter. The cloning and localization of the A2 promoter are reported here. The kinetics of RNA synthesis initiation were examined using a single-round run-off transcription assay in which multiple initiation events at a single promoter were inhibited with heparin. It was observed that the formation of heparin-resistant complexes at the A2 promoter required the presence of the initiating nucleotides, while the RNA polymerase alone was able to form heparin-resistant, non-initiated complexes at the G2 promoter. The G2 promoter was also shown by a competition assay to be a stronger promoter than A2. The effect of the delta subunit on complex formation at the two promoters was investigated with the single-round transcription assay. Delta had no effect on the formation of initiation complexes at the G2 promoter but lowered the rate and extent of complex formation at the A2 promoter. The effect of delta on the kinetic parameters of complex formation at the A2 promoter was also investigated. The data suggested that delta affects the efficiency with which the enzyme/promoter complexes undergo the transition(s) to a complex from which RNA synthesis can be initiated, although other interpretations were possible. A model for the effect of delta is proposed, in which it is postulated that the release of delta from the enzyme/promoter complex is essential for initiation. Enzyme which is associated with delta can interact with both the A2 and G2 promoters but complexes at the weaker A2 promoter do not efficiently release delta, thus slowing the formation of initiation complexes.

Text

2010-07-29

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http://hdl.handle.net/2429/27000

Microbiology and Immunology

University of British Columbia

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