Absolute requirement of cyclic nucleotide in the activation of the G141Q mutant cAMP receptor protein from Escherichia coli.
cAMP receptor protein (CRP), when interacting with cAMP, controls the expression of a network of catabolite-sensitive genes in Escherichia coli. To understand the molecular events that lead to the activation of CRP, a combined approach of site-directed mutagenesis and thermodynamic analysis was employed to study a member of a specific class of CRP mutant, CRP, which activates the in vivo expression of CRP-dependent operons in cya- strains in the absence of exogenous cAMP. Results from in vitro studies show that the CRP mutant G141Q absolutely requires cAMP for interacting with specific DNA. A quantitative comparison of the thermodynamic parameters governing ligand binding and DNA-protein complex formation in the presence of different cyclic nucleotides leads to the conclusion that this CRP mutant is activated only in the presence of cyclic nucleotides. The specificity toward cyclic nucleotides exhibited by wild-type CRP is lost in this mutant. Furthermore, the binding affinity of the ligand for the first binding site of the mutant is essentially the same as that of wild-type CRP regardless of the identity of the cyclic nucleotide. Hence, the observed in vivo activation of CRP-dependent operons by G141Q in the absence of exogenous cAMP is most likely the consequence of the replacement of cAMP by other cyclic nucleotides to activate the mutant. It is also possible that trace levels of cAMP present in the cya- strain could account for the in vivo activation of the mutant. Furthermore, these results indicate that this CRP mutant does not assume the activated conformation in the absence of cyclic nucleotides, in contrast to the current model derived from results of in vivo studies.
Cheng
X
X
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-1055.
Lee
J C
JC
D001483:Base Sequence; D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism*; D003563:Cyclic CMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides
D002373:Cyclic AMP Receptor Protein; D009838:Oligodeoxyribonucleotides; D003563:Cyclic CMP; D000242:Cyclic AMP; D006152:Cyclic GMP