THE ROLE OF 2 SURFACE EXPOSED LOOPS IN TRANSCRIPTION ACTIVATION BY THE ESCHERICHIA-COLI CRP AND FNR PROTEINS

We have investigated a number of mutations that alter the ability of the the E.coli transcription factors CRP and FNR to activate transcription. In CRP, some mutations at position 159 (H159L, H159I and DELTA-159) prevent transcription activation at a number of naturally-occurring and semi-synthetic CRP-dependent promoters. We suggest that some feature of the surface-exposed turn around residue 159 is recognised by RNA polymerase during transcription activation at these promoters. Mutations at position 52 increase CRP activity and reverse the effects of H159L and DELTA-159, most likely by creating a new contact with RNA polymerase. However this new contact only gives increased expression when the CRP binding site is located 41 1/2 base pairs upstream of the transcription start site and fails to reverse the effects of H159L and DELTA-159 at promoters where the CRP site is located further upstream. To explain our results we propose that the two surface-exposed turns around residues 52 and 159 contain elements that are potential RNA polymerase docking sites: in the CRP dimer these two active patches are located on adjacent faces of different subunits. FNR, a related transcription activator, contains amino acid sequences homologous to the CRP sequence around position 52. Mutations in this zone (from residues 81 - 88 in FNR) reduce expression from an FNR-dependent promoter without stopping FNR binding to its target. This defines a patch on FNR, which is homologous to the CRP surface-exposed loop around position 52, which is involved in transcription activation, most likely by contacting RNA polymerase.