Allosteric mechanism of induction of CytR-regulated gene expression. Cytr repressor-cytidine interaction.
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.
Barbier
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Molecular Sciences, Glaxo Welcome, Research Triangle Park, North Carolina 27709, USA.
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Senear
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D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000378:metabolism*; D003562:Cytidine / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005786:Gene Expression Regulation; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D009876:Operon; D010641:Phenotype; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; C048425:CytR protein, E coli; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D003562:Cytidine