Quantitative DNase Footprint Titration: A Method for Studying Protein-DNA Interactions

Michael Brenowitz, Donald F. Senear, Madeline A. Shea, Gary K. Ackers

Research output: Contribution to journalArticle

362 Scopus citations

Abstract

This chapter discusses that individual-site binding isotherms are uniquely suited to permit the resolution of interaction parameters for systems exhibiting cooperative interactions between multiple sites. The analysis is completely general. Any number of specific sites can be analyzed regardless of the nature of the cooperative or anticooperative interactions among them. The development of the footprint titration method, which permits resolution of individual-site isotherms, permits quantitative characterization of systems that act as critical regulators of gene transcription. The thermodynamic parameters that are resolved from footprint titration can be used in two important ways to further the understanding of gene regulation. First, the binding affinities of the various components of a gene regulatory system can be used to deduce the mechanism of the regulation—for example, the successful modeling of the switch from the lysogenic-to-lytic growth stage of the lambda phage. Second, the range of precisely controlled experimental conditions over which the technique is applicable allows to measure other thermodynamic parameters—for example, enthalpies and entropies—to study the roles of the various noncovalent forces of interaction involved in protein-DNA binding and site recognition.

Original languageEnglish (US)
Pages (from-to)132-181
Number of pages50
JournalMethods in Enzymology
Volume130
Issue numberC
DOIs
StatePublished - Jan 1 1986
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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