Location of the C-Terminal Domain of the RNA Polymerase {alpha} Subunit in Different Open Complexes at the Escherichia Coli Galactose Operon Regulatory Region

Hydroxyl radical footprinting has been used to study different open complexes between Escherichia coli</it> RNA polymerase and the galactose operon regulatory region, which contains two overlapping promoters, P1</it> and P2</it>. Complexes at P1</it> were studied by exploiting a P2</it>- mutant and complexes at P2</it> were studied with a P1- mutant. We have identified the precise location of a binding in both binary RNA polymerase-galP1</it> and RNA polymerase-P2 complexes from the effects of deletion of the C-terminal domain of the RNA polymerase α subunit: α binds to different sites at the upstream end of each complex. Transcription initiation at galP1</it> can be activated by the cyclic AMP receptor protein (CRP). Addition of CRP to the RNA polymerase-galP1</it> complex displaces the C-terminal domain of α, which then binds to a different site upstream of CRP in the ternary CRP-RNA polymerase-galP1</it> complex. Thus, the C-terminal domain of α can occupy three different sites at the gal</it> operon regulatory region. We have also examined the effect of disrupting the Activating Region of CRP on interactions between CRP and the C-terminal domain of α in ternary CRP-RNA polymerase-galP1</it> complexes. Footprinting experiments show that these substitutions interfere with the contact between CRP and α but do not affect the position of α binding to its site upstream of bound CRP.