Quantitative analysis of the ternary complex of RNA polymerase, cyclic AMP receptor protein and DNA by fluorescence anisotropy measurements

• Autorzy: Bonarek P. , Kedracka-Krok S. , Kepys B. , Wasylewski Z.
• Języki publikacji: EN

Abstrakty

EN

The in vitro formation of transcription complexes with Escherichia coli RNA polymerase was monitored using fluorescence anisotropy measurements of labeled fragments of DNA. The multicomponent system consisted of holo or core RNA polymerase (RNAP) and lac or galpromoter fragments of DNA (in different configurations), in the presence or absence of CRP activator protein (wt or mutants) with its ligand, cAMP. Values of the apparent binding constants characterizing the system were obtained, as a result of all processes taking place in the system. The interaction of the promoters with core RNAP in the absence of CRP protein was characterized by apparent binding constants of 0.67 and 1.9×106M–1 for lac166 and gal178, respectively, and could be regarded as nonspecific. The presence of wt CRP enhanced the strength of the interaction of core RNAP with the promoter, and even in the case of galpromoter it made this interaction specific (apparent binding constant 2.93×107M–1). Holo RNAP bound the promoters significantly more strongly than core RNAP did (apparent binding constants 1.46 and 40.14×106M–1 for lac166 and gal178, respectively), and the presence of CRP also enhanced the strength of these interactions. The mutation in activator region 1 of CRP did not cause any significant disturbances in the holo RNAP–lacpromoter interaction, but mutation in activator region 2 of the activator protein substantially weakened the RNAP–galpromoter interaction.

Słowa kluczowe

PL

RNA polymerase; cAMP receptor protein; DNA; fluorescence anisotropy; Escherichia coli; DNA sequence; DNA fragment; protein expression; purification; binding parameter; transcription complex formation

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2008
• Tom: 55
• Numer: 3
• Opis fizyczny: p.537-547,fig.,ref.

Twórcy

autor

Bonarek P.

• Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland

autor

Kedracka-Krok S.

autor

Kepys B.

autor

Wasylewski Z.

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