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Effect of An tracts within the UP element proximal subsite of a model promoter on kinetics of open complex formation by Escherichia coli RNA polymerase.

100%

Kolasa I. K., Łoziński T., Wierzchowski K. L.

2002

vol. 49

issue 3

659-669

In the open transcription complex (RPo), Escherichia coli RNA polymerase s70 and α subunits are known to be in contact with each other and with the promoter region overlapping the -35 hexamer and the proximal part of the UP element. To probe the effect of An DNA bending tracts in this region on initiation of transcription, kinetics of the formation of RPo by Escherichia coli RNA polymerase at two groups of synthetic consensus-like promoters bearing single DNA bending tracts (i) A5 within the proximal subsite region of the UP element (promoters Pk and Pl) and (ii) A5 (Pg) or A8 (Pm) in the region including the downstream end of the proximal UP subsite and the -35 consensus hexamer was studied in vitro using the fluorescence-detected abortive initiation assay. The kinetic data obtained demonstrate that the overall second-order rate constant ka of RPo formation is: (i) by almost one order of magnitude larger at Pk and Pl, relative to that at a control unbent promoter, and mainly due to a higher value of the equilibrium constant, K1, of the initial closed complex; and (ii) several-fold smaller at Pg and Pm owing to a strongly decreased value of K1. For Pm, the latter parameter was found to be dependent exponentially on four Mg2+ ions, as compared with the seven ions remaining in equilibrium with the initial closed complex at the parent Pa promoter. This indicates that promoter region bearing a stiff A8·T8 fragment of B'-DNA forms a smaller number of ionic contacts with the α subunit. These findings provide a new insight to and support the present model of interactions between RNA polymerase α and s70 subunits with the proximal UP subsite and the -35 region of promoters.

Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

86%

Kolasa I. K., Łoziński T., Wierzchowski K. L.

2003

vol. 50

issue 4

909-920

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase α subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of σ-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

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2 Acta Biochimica Polonica

1 2003

1 2002

2 Kolasa I. K.

2 Wierzchowski K. L.

2 Łoziński T.

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Effect of An tracts within the UP element proximal subsite of a model promoter on kinetics of open complex formation by Escherichia coli RNA polymerase.

Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.