Position 127 amino acid substitutions affect the formation of CRP : cAMP :lacP complexes but not CRP : cAMP : RNA polymerase complexes at lacP

The lacP DNA binding and activation characteristics of CRP having amino aci d substitutions at position 127 were investigated. Wild-type (WT) and T127C CRP footprinted lacP DNA in the presence of DNase I in a cAMP-dependent ma nner. The T127G, T127I, and T127S forms of CRP failed to footprint lacP bot h in the absence and in the presence of cAMP. Consistent with these data, W T and T127C CRP: cAMP complexes exhibited high affinity for the lacP CRP si te whereas T127G, T127I, or T127S CRP: cAMP complexes exhibited low affinit y for the lacP CRP site. CRP:cAMP:RNA polymerase (RNAP) complexes formed at lacP in reactions that contained WT, T127C, T127G, T127I, or T127S CRP. Th ese results demonstrate that allosteric changes important for cAMP-mediated CRP activation are differentially affected by amino acid substitution at p osition 127. Proper cAMP-mediated reorientation of the DNA binding helices required either threonine or cysteine at position 127. However, cAMP-depend ent interaction of CRP with RNAP was accomplished regardless of the amino a cid at position 127. RNAP:lacP complexes that supported high-level lac RNA synthesis formed rapidly in reactions that contained WT or T127C CRP wherea s RNAP:lacP complexes that supported only low-level lac RNA synthesis forme d at slower rates in reactions that contained T127I or T127S CRP. The T127G CRP:cAMP:RNAP:lacP complex failed to activate lacP. The results of this st udy lead us to conclude that threonine 127 plays an important role in trans duction of the signal from the CRP cyclic nucleotide binding pocket that pr omotes proper orientation of the DNA binding helices and only a minor, if a ny, role in the functional exposure of the CRP RNAP interaction domain.