Binding affinity of Escherichia coli RNA polymerase*sigma54 holoenzyme for the glnAp2, nifH and nifL promoters.
Escherichia coli RNA polymerase associated with the sigma54 factor (RNAP*sigma54) is a holoenzyme form that transcribes a special class of promoters not recognized by the standard RNA polymerase*sigma70 com plex. Promoters for RNAP*sigma54 vary in their overall 'strength' and show differences in their response to the presence of DNA curvature between enhancer and promoter. In order to examine whether these effects are related to the promoter affinity, we have determined the equilibrium dissociation constant K(d) for the binding of RNAP*sigma54 to the three promoters glnAp2, nifH and nifL. Binding studies were conducted by monitoring the changes in fluorescence anisotropy upon titrating RNAP*sigma54 to carboxyrhodamine-labeled DNA duplexes. For the glnAp2 and nifH promoters similar values of K(d) = 0.94 +/- 0.55 nM and K(d) = 0.85 +/- 0.30 nM were determined at physiological ionic strength, while the nifL promoter displayed a significantly weaker affinity with K(d) = 8.5 +/- 1.9 nM. The logarithmic dependence of K(d) on the ionic strength I was -Deltalog(K(d))/Deltalog(I) = 6.1 +/- 0.5 for the glnAp2, 5.2 +/- 1.2 for the nifH and 2.1 +/- 0.1 for the nifL promoter. This suggests that the polymerase can form fewer ion pairs with the nifL promoter, which would account for its weaker binding affinity.
Vogel
Sabine K
SK
Deutsches Krebsforschungszentrum, Biophysik der Makromoleküle (H0500), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
Schulz
Alexandra
A
Rippe
Karsten
K
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005454:Fluorescence Polarization / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D005974:Glutamate-Ammonia Ligase / Q000235:genetics; D020035:Holoenzymes / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics; D019347:Potassium Acetate / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D020035:Holoenzymes; D012808:Sigma Factor; C084296:nifL protein, Bacteria; C497751:rpoN protein, E coli; D010088:Oxidoreductases; C029130:nitrogenase reductase; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase; D019347:Potassium Acetate