Wrapping of flanking non-operator DNA in lac repressor-operator complexes: implications for DNA looping.
In our studies of lac repressor tetramer (T)-lac operator (O) interactions, we observed that the presence of extended regions of non-operator DNA flanking a single lac operator sequence embedded in plasmid DNA produced large and unusual cooperative and anticooperative effects on binding constants (Kobs) and their salt concentration dependences for the formation of 1:1 (TO) and especially 1:2 (TO2) complexes. To explore the origin of this striking behavior we report and analyze binding data on 1:1 (TO) and 1:2 (TO2) complexes between repressor and a single O(sym) operator embedded in 40 bp, 101 bp, and 2514 bp DNA, over very wide ranges of [salt]. We find large interrelated effects of flanking DNA length and [salt] on binding constants (K(TO)obs, K(TO2)obs) and on their [salt]-derivatives, and quantify these effects in terms of the free energy contributions of two wrapping modes, designated local and global. Both local and global wrapping of flanking DNA occur to an increasing extent as [salt] decreases. Global wrapping of plasmid-length DNA is extraordinarily dependent on [salt]. We propose that global wrapping is driven at low salt concentration by the polyelectrolyte effect, and involves a very large number (>/similar 20) of coulombic interactions between DNA phosphates and positively charged groups on lac repressor. Coulombic interactions in the global wrap must involve both the core and the second DNA-binding domain of lac repressor, and result in a complex which is looped by DNA wrapping. The non-coulombic contribution to the free energy of global wrapping is highly unfavorable ( approximately +30-50 kcal mol(-1)), which presumably results from a significant extent of DNA distortion and/or entropic constraints. We propose a structural model for global wrapping, and consider its implications for looping of intervening non-operator DNA in forming a complex between a tetrameric repressor (LacI) and one multi-operator DNA molecule in vivo and in vitro. The existence of DNA wrapping in LacI-DNA interactions motivates the proposal that most if not all DNA binding proteins may have evolved the capability to wrap and thereby organize flanking regions of DNA.
Tsodikov
O V
OV
Department of Chemistry, University of Wisconsin-Madison, 53706, USA.
Saecker
R M
RM
Melcher
S E
SE
Levandoski
M M
MM
Frank
D E
DE
Capp
M W
MW
Record
M T
MT
D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007763:Lac Operon; D056968:Lac Repressors; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D012097:Repressor Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012097:Repressor Proteins; D004247:DNA; D011188:Potassium