LOCATION OF THE C-TERMINAL DOMAIN OF THE RNA-POLYMERASE ALPHA-SUBUNITIN DIFFERENT OPEN COMPLEXES AT THE ESCHERICHIA-COLI GALACTOSE OPERON REGULATORY REGION

Hydroxyl radical footprinting has been used to study different open co mplexes between Escherichia coil RNA polymerase and the galactose oper on regulatory region, which contains two overlapping promoters, P1 and P2. Complexes at P1 were studied by exploiting a P2(-) mutant and com plexes at P2 were studied with a P1(-) mutant, We have identified the precise location of alpha binding in both binary RNA polymerase-galP1 and RNA polymerase-P2 complexes from the effects of deletion of the C- terminal domain of the RNA polymerase alpha subunit: alpha binds to di fferent sites at the upstream end of each complex, Transcription initi ation at galP1 can be activated by the cyclic AMP receptor protein (CR P). Addition of CRP to the RNA polymerase-galP1 complex displaces the C-terminal domain of alpha, which then binds to a different site upstr eam of CRP in the ternary CRP-RNA polymerase-galP1 complex, Thus, the C-terminal domain of alpha can occupy three different sites at the gal operon regulatory region. We have also examined the effect of disrupt ing the Activating Region of CRP on interactions between CRP and the C -terminal domain of alpha in ternary CRP-RNA polymerase-galP1 complexe s. Footprinting experiments show that these substitutions interfere wi th the contact between CRP and alpha but do not affect the position of alpha binding to its site upstream of bound CRP.