In vitro characterization of cAMP receptor protein mutated at position 127
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The cyclic adenosine 3',5' monophosphate (cAMP) receptor protein (CRP) complexed with cAMP binds a region upstream of the lactose operon promoter (lacF) to facilitate RNA polymerase (RNAP) recognition of lacF. The DNA binding and lacF activation characteristics of four mutant forms of CRP were investigated. These proteins contained a single amino acid substitution (either cysteine [C], glycine [G], isoleucine [I] or serine [S]) for threonine (T) at position 127. All but the T127G CRP mediated lacF activation in the presence of cAMP. CRPicAMP-mediated lacF activity differed for the T127C, T127I and T127S forms of CRP. The level of lacF activation observed for T127C CRP was comparable to wild-type (WT) CRP. The levels mediated by the T127I CRP or the T127S CRP were about one-half that of WT CRP. Transcription reaction mixtures that contained each of these forms of CRP differed in the rate of formation of the /ACPIRNAP open complex. DNase I footprint analysis of complexes formed between lacF DNA and the position 127 CRP mutants showed the following. First, stable DNAiCRP: cAMP complexes formed only in reactions that contained WT CRP or T127C CRP. Second, stable DNA:CRP:cAMP:RNAP complexes formed in reactions that contained the WT, T127C, T127G, T127I or T127S CI. Protection of the lacF CRP-binding site by the T127G, T127I or T127S CRP was less complete than for WT or T127C CRP. Third, stable, non-specific DNAiCRP complexes formed at high CRP concentrations (in the absence of cAMP) in reactions that contained the T127C or the T127G CRP; cAMP addition produced site-specific DNA binding for T127C CRP but not for T127G CRP. Non-specific DNA binding was not observed for the WT, T127I or T127S forms of CRP. The results of this study show that position 127 amino acid substitutions in CRP differentially affect cAMP-mediated changes in CRP structure that: (1) minimize non-specific DNA affinity and introduce high affinity site-specific DNA binding in the absence of RNAP and (2) establish the productivity of CRPiRNAP interactions at lacF. Importantly, these data also show that these position 127 amino acid substitutions have little effect on CRP:RNAP:/flcP binding cooperativity.