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Abstract
The rates of association of the tetrameric Lac repressor (LacI), dimeric LacIadi (a deletion mutant of LacI), and the native dimeric Gal repressor (GalR) to DNA restriction fragments containing a single specific site were investigated using a quench-flow DNase I "footprinting" technique. The dimeric proteins, LacIadi and GalR, and tetrameric LacI possess one and two DNA binding sites, respectively. The nanomolar protein concentrations used in these studies ensured that the state of oligomerization of each protein was predominantly either dimeric or tetrameric, respectively. The bimolecular association rate constants (ka) determined for the LacI tetramer exceed those of the dimeric proteins. The values of ka obtained for LacI, LacIadi, and GalR display different dependences on [KCl]. For LacIadi and GalR, they diminish as [KCl] increases from 25 mM to 200 mM, approaching rates predicted for three-dimensional diffusion. In contrast, the ka values determined for the tetrameric LacI remain constant up to 300 mM [KCl], the highest salt concentration that could be investigated by quench-flow footprinting. The enhanced rate of association of the tetramer relative to the dimeric proteins can be modeled by enhanced "sliding" (Berg, O. G., Winter, R. B., and von Hippel, P. H. (1981) Biochemistry 20, 6929-6948) of the LacI tetramer relative to the LacIadi dimer or a combination of enhanced sliding and the superimposition of "direct transfer" mediated by the bidentate DNA interactions of the tetramer.
Highlights
DNA molecule to the specific binding sequence [2, 5,6,7,8,9]
A quench-flow DNase I “footprinting” technique [21] has been utilized to conduct kinetic studies of LacI and LacIadi at concentrations sufficient to ensure that the proteins were predominantly tetrameric and dimeric, respectively
All of the progress curves determined in these studies are adequately described by a single exponential function; no evidence of additional kinetic phases was present under any of the experimental conditions (Fig. 2B)
Summary
LacI, Lac repressor; GalR, Gal repressor; bp, base pair(s); bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. The term “sliding” describes the one-dimensional diffusion of nonspecifically bound proteins along DNA Characteristics of this mechanism are its sensitivity to monovalent ion concentration and its dependence on the length of the DNA molecule. Molecular modeling, and x-ray diffraction studies strongly suggest that GalR and LacI share similar tertiary structures [22,23,24,25,26]. These studies demonstrate that the LacI tetramer binds operator more rapidly than the LacIadi dimer. An increase in the sliding rate for the LacI tetramer relative to the LacIadi and GalR dimers is required to account fully for the differences in association rates of the proteins
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