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

• Autorzy: Kolasa I. K. , Lozinski T. , Wierzchowski K.L.
• Języki publikacji: EN

Abstrakty

EN

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 a 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 tf-sub-unit 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.

Słowa kluczowe

EN

RNA polymerase-promoter interaction; DNA binding; Escherichia coli; bacteria; RNA polymerase; open complex formation; promoter spacer region

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2003
• Tom: 50
• Numer: 4
• Opis fizyczny: p.909-920,fig.,ref.

Twórcy

autor

Kolasa I. K.

• Polish Academy of Sciences, A.Pawinskiego 5a, 02-106 Warsaw, Poland

autor

Lozinski T.

autor

Wierzchowski K.L.

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