Orientation of functional activating regions in the Escherichia coli CRP protein during transcription activation at class II promoters

At class II CRP-dependent promoters the DNA site for CRP overlaps the DNA site for RNA polymerase, covering the -35 region, Transcription activation at class II CRP-dependent promoters requires a contact between an activating region in the upstream subunit of the bound CRP dimer and a contact site in the C-terminal domain of the alpha-subunit of RNA polymerase. Transcription activation is suppressed by amino acid substitutions in the activating region, but activation can be restored by second site substitutions at K52 or E96, These substitutions identify two separate regions on the surface of CRP that appear to be able to interact with RNA polymerase specifically at class II promoters, Using the method of 'oriented heterodimers' we show that these alternative activating regions are functional in the downstream subunit of the bound CRP dimer.