'Footprint' titrations yield valid thermodynamic isotherms

M. Brenowitz, D. F. Senear, M. A. Shea, G. K. Ackers

Research output: Contribution to journalArticle

145 Scopus citations

Abstract

A central issue in gene regulation is the mechanism, and biological function, of the cooperative binding of regulatory protein ligands to specific sites on DNA. To elucidate the physical-chemical basis of these interactions we have developed a thermodynamically rigorous method for conducting DNase I 'footprint' (protection) titration experiments. The intrinsic binding constants and also those for cooperative interactions between various sites can be resolved from the individual-site binding curves determined by this technique. Experimental studies of cI-repressor-operator binding have demonstrated that the method provides an accurate representation of the fractional saturation of a binding site. We present individual-site binding curves for a λ operator with two competent sites that demonstrate the presence of cooperative interactions between the sites. These curves set a lower limit to the magnitude of the cooperative free energy without comparison to single-site mutant operators.

Original languageEnglish (US)
Pages (from-to)8462-8466
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume83
Issue number22
DOIs
StatePublished - Jan 1 1986
Externally publishedYes

ASJC Scopus subject areas

  • General

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