Thermodynamics of E. coli cytidine repressor interactions with DNA: distinct modes of binding to different operators suggests a role in differential gene regulation.
Interactions between the Escherichia coli cytidine repressor protein (CytR) and its operator sites at the different promoters that comprise the CytR regulon, play an important role in the regulation of these promoters. The natural operators are palindromes separated by variable length central spacers (0-9 bp). We have suggested that this variability affects the flexibility of CytR-DNA contacts, thereby affecting the critical protein-protein interactions between CytR and the cAMP receptor protein (CRP) that underlie differential repression and activation of CytR-regulated genes. To assess this hypothesis, we investigated the thermodynamics of CytR binding to the natural operator sequences found in udpP and deoP2. To separate effects due to spacing from effects due to the differing sequences of the recognition half-sites of these two operators, we also investigated CytR binding to artificial hybrid operators, in which the half-site sequences of udpP and deoP2 were exchanged. Thermodynamic parameters, DeltaS(o), DeltaH(o) and DeltaC(o)(p), were determined by van't Hoff analysis of CytR binding, monitored by changes in the steady-state fluorescence anisotropy of dye-conjugated, operator-containing oligonucleotides. Large differences in thermodynamics were observed that depend primarily on the central spacer rather than the sequences of the recognition half-sites. Binding to operators with deoP2 spacing results in a very large, negative DeltaC(o)(p). Association is strongly favored enthalpically and strongly disfavored entropically at ambient temperature. By contrast, binding to operators with udpP spacing results in a small, negative DeltaC(o)(p). Association is weakly favored both enthalpically and entropically at ambient temperature. A difference of such magnitude in DeltaDeltaC(o)(p) has not been reported previously for specific binding of a transcription factor to different sites. The identical salt dependence of CytR binding to deoP2 and udpP operators indicates that ion-dependent processes do not contribute significantly to this difference. Thus, the different thermodynamic effects appear to reflect distinctly different modes of site-specific DNA binding. We discuss similarities to operator binding by CytR homologs among LacI family repressors, and we consider how different CytR binding modes might affect interactions with other components of the gene regulatory machinery that contribute to differential gene regulation.
Tretyachenko-Ladokhina
Vira
V
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA.
Ross
J B Alexander
JB
Senear
Donald F
DF
D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005454:Fluorescence Polarization; D015964:Gene Expression Regulation, Bacterial; D008957:Models, Genetic; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D009875:Operator Regions, Genetic / Q000235:genetics*; D009994:Osmolar Concentration; D011485:Protein Binding / Q000187:drug effects; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D012492:Salts / Q000494:pharmacology; D013379:Substrate Specificity; D013816:Thermodynamics
C048425:CytR protein, E coli; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D012097:Repressor Proteins; D012492:Salts