Comparison of the DNA association kinetics of the Lac repressor tetramer, its dimeric mutant LacIadi, and the native dimeric Gal repressor.
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.
Hsieh
M
M
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Brenowitz
M
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry; D019281:Dimerization; D029968:Escherichia coli Proteins; D007700:Kinetics; D056968:Lac Repressors; D011487:Protein Conformation; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C050993:Galactose repressor proteins; D056968:Lac Repressors; D012097:Repressor Proteins; D004247:DNA