Study of Highly Constitutively Active Mutants Suggests How cAMP Activates cAMP Receptor Protein

Abstract

and Ser 128 and (ii) the concomitant reorientation of the ␤4/␤5 loop. Finally, we also report that elevated expression of T127L/S128I CRP markedly perturbed E. coli growth even in the absence of cAMP, which suggests why comparably active variants have not been described previously.

Functional Analysis of the Carboxy-Terminal Region of Bacillus subtilis TnrA, a MerR Family Protein

“…This aberrant gene expression may be the cause of the growth impairment observed on minimal media with the class II mutants. Interestingly, a similar phenomenon has been observed for Escherichia coli, where highly constitutive mutants of the cAMP activator protein have been shown to inhibit cell growth (51). Nonetheless, the growth phenotype of the class II mutants is puzzling in that strains containing a null mutation in glnA, the gene encoding GS, are able to grow on glutamine minimal media even though TnrA is constitutively active (45).…”

Two-State Allosteric Modeling Suggests Protein Equilibrium as an Integral Component for Cyclic AMP (cAMP) Specificity in the cAMP Receptor Protein ofEscherichia coli

“…CRP is a homodimer in which each subunit contains two domains, the cAMP-binding domain and the DNA-binding domain, separated by a hinge region (27). The structure of the inactive form of CRP has never been determined, so the mechanism of allosteric activation by cAMP is conjectural, but plausible models for the conformational change that cAMP effects have been proposed (29,39).…”

“…CRP is a homodimer in which each subunit contains two domains, the cAMP-binding domain and the DNA-binding domain, separated by a hinge region (27). The structure of the inactive form of CRP has never been determined, so the mechanism of allosteric activation by cAMP is conjectural, but plausible models for the conformational change that cAMP effects have been proposed (29,39).CRP variants have been found that have altered ligand specificity (1,5,9,14,19,23,40). The substitutions in some of these variants lie in the effector-binding pocket itself and presumably alter the specific interactions between CRP and the ligand (23, 40).…”

“…E. coli crp was cloned into pEXT20, and then six histidine codons were subsequently added at the 3Ј end as described previously (39). WT CRP and CRP variants used in the present study were all His tagged.…”

“…Codon randomization involved similar PCR amplification as described above, but the primers contained completely randomized codons at the desired positions. For screening CRP* activity in vivo, we used a cya crp E. coli strain, UQ3811 (39) with lacZ under the control of the class I CC(Ϫ61.5) promoter, and monitored the ␤-galactosidase accumulation in colonies. The crp genes from selected variants were sequenced to determine the causative residue changes.…”