COMPARISON OF THE DNA ASSOCIATION KINETICS OF THE LAC REPRESSOR TETRAMER, ITS DIMERIC MUTANT LACI(ADI) AND THE NATIVE DIMERIC GAL REPRESSOR

The rates of association of the tetrameric Lac repressor (LacI), dimer ic LacI(adi) (a deletion mutant of LacI), and the native dimeric Gal r epressor (GalR) to DNA restriction fragments containing a single speci fic site were investigated using a quench-flow DNase I ''foot printing '' technique, The dimeric proteins, LacI(adi) and GalR, and tetrameric LacI possess one and two DNA binding sites, respectively, The nanomol ar protein concentrations used in these studies ensured that the state of oligomerization of each protein was predominantly either dimeric o r tetrameric, respectively, The bimolecular association rate constants (k(a)) determined for the LacI tetramer exceed those of the dimeric p roteins, The values of k(a) obtained for LacI, LacI(adi), and GalR dis play different dependences on [KCl], For LacI(adi) and GalR, they dimi nish as [KCl] increases from 25 mM to 200 mM, approaching rates predic ted for three-dimensional diffusion, In contrast, the k(a) values dete rmined for the tetrameric LacI remain constant up to 300 mM [KCl] the highest salt concentration that could be investigated by quench-flow f ootprinting. 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, 69 29-6948) of the LacI tetramer relative to the LacI(adi) dimer or a com bination of enhanced sliding and the superimposition of ''direct trans fer'' mediated by the bidentate DNA interactions of the tetramer.