title: Role of protein-protein bridging interactions on cooperative assembly of DNA-bound CRP-CytR-CRP complex and regulation of the Escherichia coli CytR regulon.
abstract: The unlinked operons that comprise the Escherichia coli CytR regulon are controlled coordinately through interactions between two gene regulatory proteins, the cAMP receptor protein (CRP) and the cytidine repressor (CytR). CytR controls the balance between CRP-mediated recruitment and activation of RNA polymerase and transcriptional repression. Cooperative interactions between CytR, when bound to an operator (CytO) located upstream of a CytR-regulated promoter, and CRP, when bound to flanking tandem promoters, are critical to the regulatory role of CytR. When CytR binds cytidine, cooperativity is reduced resulting in increased transcriptional activity. However, this cytidine-mediated effect varies among promoters, suggesting that coupling between cytidine binding to CytR and CytR-CRP association is sensitive to promoter structure. To investigate the chemical and structural basis for these effects, we investigated how cytidine binding affects association between CytR and CRP in solution and how it affects the binding of CytR deletion mutants lacking the DNA binding HTH domain, with tandem CRP dimers bound to either udpP or deoP2. Deletion mutants that, as we show here, retain the native functions of the allosteric, inducer-binding domain but do not bind DNA were expressed and purified. We refer to these as Core domain. Despite only weak association between CytR and CRP in solution, our results demonstrate the formation of a relatively stable complex in which the Core domain forms a protein bridge between tandem CRP dimers when bound to either udpP or deoP2. The DeltaG(o) for bridge complex formation is about -7.8 kcal/mol. This is well in excess of that required to account for cooperativity (-2.5 to -3 kcal/mol). The bridge complexes are significantly destabilized by cytidine binding, and to the same extent in both promoter complexes (DeltaDeltaG(o) approximately +2 kcal/mol). Even with this destabilization, DeltaG(o) for bridge complex formation by cytidine-liganded Core domain is still sufficient by itself to account for cooperativity. These findings demonstrate that direct coupling between cytidine binding to CytR and CytR-CRP association does not account for promoter-specific effects on cooperativity. Instead, cytidine binding must induce a CytR conformation that is more rigid or in some other way less tolerant of the variation in the geometric arrangement of operator sites between different promoters.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, USA.
authors.forename: Mayy
authors.initials: M
authors.lastname: Chahla
authors.forename: John
authors.initials: J
authors.lastname: Wooll
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Laue
authors.forename: Nghia
authors.initials: N
authors.lastname: Nguyen
authors.forename: Donald F
authors.initials: DF
authors.lastname: Senear
mesh_terms: D001665:Binding Sites; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism*; D003562:Cytidine / Q000378:metabolism; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D018085:Regulon / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014543:Uridine Phosphorylase / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; C048425:CytR protein, E coli; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D003562:Cytidine; D014543:Uridine Phosphorylase; D012321:DNA-Directed RNA Polymerases