Evidence for Contact between the Cyclic AMP Receptor Protein and the σ70 Subunit of Escherichia coli RNA Polymerase

Abstract

The loop at the 52-position of the cAMP receptor protein (CRP) has been suggested as a potential site for contacting RNA polymerase on Class II promoters where the CRP binding site is located at position -41.5 (Bell, A., Gaston, K., Williams, R., Chapman, K., Kolb, A., Buc, H., Minchin, S., Williams, J., and Busby, S. (1990) Nucleic Acids Res. 18, 7243-7250). Using protein-protein photo-cross-linking, evidence is presented showing that the 52-loop of CRP is in physical proximity to the delta subunit of RNA polymerase holoenzyme. This interaction required the presence of a functional preinitiation complex. The CRP suppressor mutation, K52N, increased the efficiency of cross-linking, indicating an improved physical interaction between the CRP 52-loop and the delta subunit. Evidence for direct interaction between the CRP 156-162 loop and delta subunit of RNA polymerase on both gal and lac promoters are also provided. The data indicate that CRP bound to the gal promoter contacts both the alpha and delta 70 subunits of RNA polymerase.