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Free keywords:
Kinetics, DNA binding, Bending, Stopped flow, Electric dichroism, Pseudo-sites
Abstract:
The kinetics of cAMP receptor (CAP) binding to promoter DNA has been studied by stopped-flow electric-dichroism at a reduced salt concentration, where the coupling of non-specific and specific binding can be observed directly. Amplitudes, rise and decay times of dichroism transients provide detailed information about the reaction and the structure of intermediates over more than six orders of magnitude on the time scale. CAP binding during the first milliseconds after mixing is indicated by an increase of both rise- and decay-time constants. A particularly large increase of rise times reflects initial formation of non-symmetric complexes by protein binding to non-specific sites at DNA ends. The increase of the hydrodynamic dimensions continues up to ~1 s, before a decrease of time constants reflects transition to compact states with bent DNA up to the time range of ~103 s. The slow approach to CAP-induced DNA bending is due to non-specific complexes, which are formed initially and are converted slowly to the specific complex. At the salt concentration of 13.5 mM, conversion to specific complexes with bent DNA is completed after ~40 s at pH 8 compared to >103 s at pH 7, resulting from a higher affinity of CAP to non-specific sites at pH 7 than 8 by a factor of ~100. Thus, under the given conditions non-specific sites delay rather than facilitate formation of the specific complex with bent DNA. Experimental data obtained for a non-specific DNA clearly indicate the impact of pseudo-sites. The different electro-optical parameters have been combined in global fits.
