Comparison of the DNA association kinetics of the Lacy repressor tetramet, its dimeric mutant LacI(adi), and the native dimeric gal repressor

Mark Hsieh, Michael Brenowitz

Research output: Contribution to journalArticle

39 Scopus citations

Abstract

The rates of association of the tetrameric Lacy repressor (LacI), dimeric LacI(adi) (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 'foot-printing' technique. The dimeric proteins, LacI(adi) 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 (k(a)) determined for the LacI tetramer exceed those of the dimeric proteins. The values of k(a) obtained for LacI, LacI(adi), and GalR display different dependences on [KCl]. For LacI(adi) and GalR, they diminish as [KCl] increases from 25 mM to 200 mM, approaching rates predicted for three-dimensional diffusion. In contrast, the k(a) 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 LaeI(adi) dimer or a combination of enhanced sliding and the superimposition of 'direct transfer' mediated by the bidentate DNA interactions of the tetramer.

Original languageEnglish (US)
Pages (from-to)22092-22096
Number of pages5
JournalJournal of Biological Chemistry
Volume272
Issue number35
DOIs
StatePublished - Aug 29 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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