Nucleic Acids Research, 2002, Vol. 30, No. 18 4094-4101
© 2002 Oxford University Press
Binding affinity of Escherichia coli RNA polymerase·
54 holoenzyme for the glnAp2, nifH and nifL promoters
Deutsches Krebsforschungszentrum, Biophysik der Makromoleküle (H0500), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany and 1 Deutsches Krebsforschungszentrum, Molekulare Genetik (H0700), Im Neuenheimer Feld 280 and Kirchhoff-Institut für Physik, Physik molekularbiologischer Prozesse, Universität Heidelberg, Schröderstraße 90, D-69120 Heidelberg, Germany
*To whom correspondence should be addressed. Tel: +49 6221 424620; Fax: +49 6221 42524676; Email: karsten.rippe{at}dkfz.de
Present address:
Alexandra Schulz, Roche Diagnostics, Sandhoferstraße 116, D-68305 Mannheim, Germany
Escherichia coli RNA polymerase associated with the 
54 factor (RNAP·54) is a holoenzyme form that transcribes a special class of promoters not recognized by the standard RNA polymerase·70 com plex. Promoters for RNAP·54 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 Kd for the binding of RNAP·54 to the three promoters glnAp2, nifH and nifL. Binding studies were conducted by monitoring the changes in fluorescence anisotropy upon titrating RNAP·54 to carboxyrhodamine-labeled DNA duplexes. For the glnAp2 and nifH promoters similar values of Kd = 0.94 ± 0.55 nM and Kd = 0.85 ± 0.30 nM were determined at physiological ionic strength, while the nifL promoter displayed a significantly weaker affinity with Kd = 8.5 ± 1.9 nM. The logarithmic dependence of Kd on the ionic strength I was –
log(Kd)/log(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.
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