Structures during binding of cAMP receptor to promoter DNA: promoter search slowed by non-specific 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 ~10(3) 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 >10(3) 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.

Allosteric Control of cAMP Receptor Binding Dynamics

“…Thus, it is likely that the effects recorded for the consensus DNA also mainly reflect binding to nonspecific sites, resulting from the fact that nonspecific sites are abundant. Conversion of nonspecific to specific complexes is expected in the case of consensus DNA, but it is hardly possible to assign any of the observed components in the fluorescence transients to such conversion, unless additional information, e.g., by stopped-flow electrooptics, 17 is used.…”

“…The sequence of ns-40bp was selected for its minimal content of segments corresponding to pseudosites. 17 The following buffers were used: 5 mM NaCl, 10 mM sodium cacodylate pH 7, and 0.1 mM EDTA/DTT (buffer A); 10 mM NaCl, 5 mM Tris pH 8.0, and 0.1 mM EDTA/DTT (buffer B); 90 mM NaCl, 10 mM sodium cacodylate pH 7, and 0.1 mM EDTA/DTT (buffer C); 90 mM NaCl, 20 mM Tris pH 8.0, and 0.1 mM EDTA/DTT (buffer D).…”

“…The single strands for a nonspecific DNA double helix with 40 bp (ns-40bp, sequence GCGTCCAGTT CGTTGAGCTT CTCCAGCAGC GTTAATGTCG) were synthesized and annealed by the same procedure. The sequence of ns-40bp was selected for its minimal content of segments corresponding to pseudosites …”

“…The electro-optical data demonstrate that the broad time range for formation of the specific complex with bent DNA at low salt is due to a slow transfer of the protein from nonspecific sites to the specific site. 17 This delay effect is reduced when the lifetime of nonspecific complexes is decreased. By this argument, it may be expected that specific complexes are formed at a high rate, when the salt concentration is high.…”

“…The information about the dynamics of the CAP system is not as advanced yet, but some results − obtained by various approaches have been presented. In a recent study based on stopped-flow electro-optics, a rather complex sequence of reactions was detected for the binding of CAP to promoter DNA at a reduced salt concentration of 13.5 mM. For technical reasons, stopped-flow electro-optics could not be applied at physiological salt concentrations.…”