Abstract
Transcription from cistrons of the Escherichia coli CytR regulon is activated by E. coli cAMP receptor protein (CRP) and repressed by a multiprotein complex composed of CRP and CytR. De-repression results when CytR binds cytidine. CytR is a homodimer and a LacI family member. A central question for all LacI family proteins concerns the allosteric mechanism that couples ligand binding to the protein-DNA and protein-protein interactions that regulate transcription. To explore this mechanism for CytR, we analyzed nucleoside binding in vitro and its coupling to cooperative CytR binding to operator DNA. Analysis of the thermodynamic linkage between sequential cytidine binding to dimeric CytR and cooperative binding of CytR to deoP2 indicates that de-repression results from just one of the two cytidine binding steps. To test this conclusion in vivo, CytR mutants that have wild-type repressor function but are cytidine induction-deficient (CID) were identified. Each has a substitution for Asp281 or neighboring residue. CID CytR281N was found to bind cytidine with three orders of magnitude lower affinity than wild-type CytR. Other CytR mutants that do not exhibit the CID phenotype were found to bind cytidine with affinity similar to wild-type CytR. The rate of transcription regulated by heterodimeric CytR composed of one CytR281N and one wild-type subunit was compared with that regulated by wild-type CytR under inducing conditions. The data support the conclusion that the first cytidine binding step alone is sufficient to induce.
Highlights
Transcription from cistrons of the Escherichia coli CytR regulon is activated by E. coli cAMP receptor protein (CRP) and repressed by a multiprotein complex composed of CRP and CytR
The transport proteins and enzymes required for nucleoside utilization in Escherichia coli are encoded by genes belonging to the CytR regulon [1]
The critical role of CytRzCRP cooperativity is highlighted by the mechanism of cytidine-mediated induction
Summary
Transcription from cistrons of the Escherichia coli CytR regulon is activated by E. coli cAMP receptor protein (CRP) and repressed by a multiprotein complex composed of CRP and CytR. The basic DNA binding unit of each of these proteins is a homodimer in which helix-turn-helix domains from both subunits combine to form the DNA binding interface Since both subunits harbor identical ligand binding sites, the allosteric mechanism that couples inducer or corepressor binding to changes in the macromolecular interactions that regulate transcription is an important issue to this entire family of proteins. For both PurR and LacI, conformational transitions that accompany ligand binding have been investigated by x-ray crystallography (4 – 6). While the structural mechanisms that couple ligand binding to tertiary conformation differ in the two proteins (4 – 6), the tertiary and quarternary structural perturbations are remarkably similar
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