CRP Binding and Transcription Activation at CRP-S Sites

Abstract

In Haemophilus influenzae, as in Escherichia coli, the cAMP receptor protein (CRP) activates transcription from hundreds of promoters by binding symmetrical DNA sites with the consensus half-site 5′-A 1A 2A 3T 4G 5T 6G 7A 8T 9C 10T 11. We have previously identified 13 H. influenzae CRP sites that differ from canonical (CRP-N) sites in the following features: (1) Both half-sites of these noncanonical (CRP-S) sites have C 6 instead of T 6, although they otherwise have an unusually high level of identity with the binding site consensus. (2) Only promoters with CRP-S sites require both the CRP and Sxy proteins for transcription activation. To study the functional significance of CRP-S site sequences, we purified H. influenzae ( Hi)CRP and compared its DNA binding properties to those of the well-characterized E. coli ( Ec)CRP. All EcCRP residues that contact DNA are conserved in HiCRP, and both proteins demonstrated a similar high affinity for the CRP-N consensus sequence. However, whereas EcCRP bound specifically to CRP-S sites in vitro, HiCRP did not. By systematically substituting base pairs in native promoters and in the CRP-N consensus sequence, we confirmed that HiCRP is highly specific for the perfect core sequence T 4G 5T 6G 7A 8 and is more selective than EcCRP at other positions in CRP sites. Even though converting C 6 → T 6 greatly enhanced HiCRP binding to a CRP-S site, this had the unexpected effect of nearly abolishing promoter activity. A + T-rich sequences upstream of CRP-S sites were also found to be required for promoter activation, raising the possibility that Sxy binds these A + T sequences to simultaneously enable CRP–DNA binding and assist in RNA polymerase recruitment.