Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase

Abstract

Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein(F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 .mu.M in the presence of cyclic AMP and about 100 .mu.M in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 .mu.M) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.