Equilibrium studies of the cyclic AMP receptor protein-DNA interaction

Michael G. Fried, Donald M. Crothers

Research output: Contribution to journalArticlepeer-review

120 Scopus citations

Abstract

The binding of the Escherichia coli cyclic AMP receptor protein (CAP) to restriction fragments containing the lac promoter-operator region has been investigated as a function of cAMP concentration, using a sensitive gel electrophoresis assay. Under standard conditions (13 mM ionic strength), the equilibrium constant for CAP binding to its primary site on a 203 base-pair lac promoter fragment is 6·3×108M-1 at 0·2 μM-cAMP, and increases to 8·4×1010 M-1 at 5·0 μM-cAMP. The latter is about 105 times larger than the equilibrium constant for binding to an isolated, non-specific site. The L8 mutation, which renders the lac promoter unresponsive to CAP in vivo, lowers this binding affinity by five- to tenfold. Analysis of the cAMP dependency of binding over the concentration range of 0·2 μM to 10 μM reveals that uptake of a single equivalent of cAMP is required for site-specific binding. Similarly, the transfer of CAP from a non-specific DNA site to a specific site requires the net uptake of a single molecule of cAMP. In contrast, co-operative non-specific binding to DNA was found to be independent of cAMP concentration with an equilibrium binding constant of 6×106 M-1. We conclude that the cAMP affinity of the two CAP subunits in the specific promoter complex is not equal, and that the complex structure therefore deviates significantly from twofold symmetry. A model for the regulation of the lac promoter by the intracellular cAMP concentration is proposed on the basis of the equilibrium binding results.

Original languageEnglish
Pages (from-to)241-262
Number of pages22
JournalJournal of Molecular Biology
Volume172
Issue number3
DOIs
StatePublished - Jan 25 1984

Bibliographical note

Funding Information:
This work was supported by a grant GM 21966 from the National Institutes of Health. We thank Peter vonHippel for helpful comments on:the manuscript.

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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