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• W2007127972 abstract "The CytR repressor fulfills dual roles as both a repressor of transcription from promoters of the Escherichia coli CytR regulon and a co-activator in some circumstances. Transcription is repressed by a three-protein complex (cAMP receptor protein (CRP)-CytR-CRP) that is stabilized by cooperative interactions between CRP and CytR. However, cooperativity also means that CytR can recruit CRP and, by doing so, can act as a co-activator. The central role of cooperativity in regulation is highlighted by the fact that binding of the inducer, cytidine, to CytR is coupled to CytR-CRP cooperativity; this underlies the mechanism for induction. Similar interactions at the different promoters of the CytR regulon coordinate expression of the transport proteins and enzymes required for nucleoside catabolism but also provide differential expression of these genes. A fundamental question in both prokaryotic and eukaryotic gene regulation is how combinatorial mechanisms of this sort regulate differential expression. Recently, we showed that CytR binds specifically to multiple sites in the E. coli deoPpromoter, thereby providing competition for CRP binding to CRP operator site 1 (CRP1) and CRP2 as well as cooperativity. The effect of the competition at this promoter is to negate the role of CytR in recruiting CRP. Here, we have used quantitative footprint and mobility shift analysis to investigate CRP and CytR binding to theE. coli udp promoter. Here too, we find that CytR both cooperates and competes for CRP binding. However, consistent with both the distribution of CytR recognition motifs in the sequence of the promoter and the regulation of the promoter, the competition is limited to CRP2. When cytidine binds to CytR, the effect on cooperativity is very different at the udp promoter than at thedeoP2 promoter. Cooperativity with CRP at CRP1 is nearly eliminated, but the effect on CytR-CRP2 cooperativity is negligible. These results are discussed in relation to the current structural model of CytR in which the core, inducer-binding domain is tethered to the helix-turn-helix, DNA-binding domain via flexible peptide linkers. The CytR repressor fulfills dual roles as both a repressor of transcription from promoters of the Escherichia coli CytR regulon and a co-activator in some circumstances. Transcription is repressed by a three-protein complex (cAMP receptor protein (CRP)-CytR-CRP) that is stabilized by cooperative interactions between CRP and CytR. However, cooperativity also means that CytR can recruit CRP and, by doing so, can act as a co-activator. The central role of cooperativity in regulation is highlighted by the fact that binding of the inducer, cytidine, to CytR is coupled to CytR-CRP cooperativity; this underlies the mechanism for induction. Similar interactions at the different promoters of the CytR regulon coordinate expression of the transport proteins and enzymes required for nucleoside catabolism but also provide differential expression of these genes. A fundamental question in both prokaryotic and eukaryotic gene regulation is how combinatorial mechanisms of this sort regulate differential expression. Recently, we showed that CytR binds specifically to multiple sites in the E. coli deoPpromoter, thereby providing competition for CRP binding to CRP operator site 1 (CRP1) and CRP2 as well as cooperativity. The effect of the competition at this promoter is to negate the role of CytR in recruiting CRP. Here, we have used quantitative footprint and mobility shift analysis to investigate CRP and CytR binding to theE. coli udp promoter. Here too, we find that CytR both cooperates and competes for CRP binding. However, consistent with both the distribution of CytR recognition motifs in the sequence of the promoter and the regulation of the promoter, the competition is limited to CRP2. When cytidine binds to CytR, the effect on cooperativity is very different at the udp promoter than at thedeoP2 promoter. Cooperativity with CRP at CRP1 is nearly eliminated, but the effect on CytR-CRP2 cooperativity is negligible. These results are discussed in relation to the current structural model of CytR in which the core, inducer-binding domain is tethered to the helix-turn-helix, DNA-binding domain via flexible peptide linkers. The Escherichia coli CytR regulon comprises at least nine unlinked transcriptional units that encode enzymes and transport proteins required for nucleoside catabolism and recycling. CRP 1The abbreviations used are: CRP, cAMP receptor protein; CRP1 and CRP2, CRP operator sites 1 and 2, respectively; RNAP, E. coli RNA polymerase; bp, base pair(s); bis-Tris, bis(2-hydroxyethyl)imino- tris(hydroxymethyl)methane. activates transcription of these units in response to intracellular cAMP levels. Transcription is repressed by CytR, a member of the LacI family of bacterial repressors, and is induced when CytR binds cytidine. These features are common to all of the unlinked transcriptional units that comprise the regulon and serve together to coordinate their regulation (1Valentin-Hansen P. Sogaard-Andersen L. Pedersen H. Mol. Microbiol. 1996; 20: 461-466Crossref PubMed Scopus (95) Google Scholar). However, a key feature of the CytR regulon is that extents of activation, repression, and induction vary substantially among the different transcription units (cf. Refs. 2Martinussen J. Mollegaard N.E. Holst B. Douthwaite S.R. Valentin-Hansen P. Gralla J.D. DNA-Protein Interactions in Transcription. Alan R. Liss, New York1989: 31-41Google Scholar and 3Barbier C.S. Short S.A. Senear D.F. J. Biol. Chem. 1997; 272: 16962-16971Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). An intriguing question is how the interplay among these two transcriptional regulatory proteins and the various promoters yields differential regulation. Presumably, combinatorial mechanisms that rely on local features of the promoters, such as different arrangements of the control elements, are involved. Similar combinatorial mechanisms also appear to be important in the regulation of cell growth and differentiation, processes that also often involve a small number of key regulatory proteins. Thus, the CytR regulon has general significance as a model for understanding gene regulatory processes. Our goal is to understand how functional, multi-protein, transcription complexes form at different promoters with different arrangements of protein binding sites. CytR has two features that are not observed in other LacI family members and that appear to be important to its role as a differential transcriptional regulator. First, CytR and CRP bind cooperatively to form a three-protein complex on the DNA. In this complex, CytR binds to an operator site (usually referred to as CytO) that is flanked by tandem CRP operators, CRP1 and CRP2, as are found in most CytR-regulated promoters. CytR forms a protein bridge between the bound CRP dimers. The importance of this cooperativity is highlighted by the fact that expression is induced because the cooperativity is lost when CytR binds cytidine; cytidine binding to CytR has no effect on the intrinsic DNA binding of CytR (4Pedersen H. Sogaard-Andersen L. Holst B. Valentin-Hansen P. J. Biol. Chem. 1991; 266: 17804-17808Abstract Full Text PDF PubMed Google Scholar, 5Perini L.T. Doherty E.A. Werner E. Senear D.F. J. Biol. Chem. 1996; 271: 33242-33255Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Therefore, it is the three-protein complex that is the functional repressor, not CytR alone. Second, CytR exhibits lower DNA binding specificity than other LacI family members and most other bacterial repressors. As a consequence, as detailed below, CytR does not bind only to CytO but also binds to additional binding sites whose number and arrangement appear to differ among the promoters. Given these facts, we wish to address two questions. First, how does binding of CytR differ in different promoters? Second, how do these differences affect cooperativity and its modulation by cytidine? A key to how CytR binds to different promoters is its relatively broad DNA sequence specificity. Like other LacI family members, the basic DNA binding unit of CytR is a homodimer (6Kristensen H.H. Valentin-Hansen P. Sogaard-Andersen L. J. Mol. Biol. 1996; 260: 113-119Crossref PubMed Scopus (19) Google Scholar). As expected, based on this quarternary structure, CytR binding sites contain tandem recognition motifs. However, the exact recognition motif has proven difficult to define. It has been reported as both TGCAA (7Rasmussen P.B. Sogaard-Andersen L. Valentin-Hansen P. Nucleic Acids Res. 1993; 21: 879-885Crossref PubMed Scopus (22) Google Scholar) and, more recently, as GTTGCATT (8Pedersen H. Valentin-Hansen P. EMBO J. 1997; 16: 2108-2118Crossref PubMed Scopus (47) Google Scholar), based on different systematic evolution of ligands by exponential enrichment experiments conducted by the same group. Based on where CytR binds specifically to the CytR-regulated deoP1and deoP2 promoters, we proposed that TTGCAA, a symmetric variant of these sequences, is the recognition sequence (5Perini L.T. Doherty E.A. Werner E. Senear D.F. J. Biol. Chem. 1996; 271: 33242-33255Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). CytR is also unusually tolerant of variation in spacing between recognition motifs. The preferred spacing is 2–3 base pairs (7Rasmussen P.B. Sogaard-Andersen L. Valentin-Hansen P. Nucleic Acids Res. 1993; 21: 879-885Crossref PubMed Scopus (22) Google Scholar, 8Pedersen H. Valentin-Hansen P. EMBO J. 1997; 16: 2108-2118Crossref PubMed Scopus (47) Google Scholar), but CytR-mediated regulation of gene expression has been demonstrated on synthetic promoters in vivo with spacing up to three helical turns (9Jorgensen C.I. Kallipolitis B.H. Valentin-Hansen P. Mol. Microbiol. 1998; 27: 41-50Crossref PubMed Scopus (40) Google Scholar). In this context, it is important to note that most CytR-regulated promoters feature multiple degenerate repeats of the (T)TGCAA sequence motif with variable spacing between them. Depending upon the spacing between such repeats, additional CytR binding sites might exist at these promoters. In fact, when we investigated CytR binding to thedeoP2 promoter, we found that CytR does bind specifically to multiple sites (5Perini L.T. Doherty E.A. Werner E. Senear D.F. J. Biol. Chem. 1996; 271: 33242-33255Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). CytR and CRP bind cooperatively when CytR binds CytO. However, CytR also binds to separate, specific sites atdeoP2, one of which overlaps CRP1, and another that overlaps CRP2. In this situation, CytR competes directly for CRP binding to CRP1 and CRP2. This special mode of protein-DNA interaction in which CytR can either enhance CRP binding or compete for CRP binding affects both repression and activation. Repression by the cooperative CRP-CytR-CRP complex results from competition between CytR and RNAP, both of which are recruited by CRP to bind to the DNA sequence flanked by CRP1 and CRP2 (10Belyaeva T.A. Rhodius V.A. Webster C.L. Busby S.J. J. Mol. Biol. 1998; 277: 789-804Crossref PubMed Scopus (71) Google Scholar). However, competition between CytR and CRP for binding to CRP1 and CRP2 provides a second mode of CytR-mediated repression. In activation, one consequence of the competition between CytR and CRP is to facilitate configurations in which CRP is bound either to CRP1 or to CRP2, but not to both. This is significant because CRP1 and CRP2 are thought to mediate different mechanisms of activation (11Ushida C. Aiba H. Nucleic Acids Res. 1990; 18: 6325-6330Crossref PubMed Scopus (114) Google Scholar, 12Ebright R.H. Mol. Microbiol. 1993; 8: 797-802Crossref PubMed Scopus (168) Google Scholar). Based on their sequences (Ref. 5Perini L.T. Doherty E.A. Werner E. Senear D.F. J. Biol. Chem. 1996; 271: 33242-33255Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar; Fig. 1), we expect the different promoters to vary as to whether CytR competes for CRP binding to CRP1, to CRP2, or to both sites. The primary function of CytR may be differential modulation of CRP1-mediated versus CRP2-mediated activation. In this way, different patterns of CytR binding at different promoters might provide differential gene regulation. The broad DNA binding specificity of CytR might also result in different contributions to the stability of the three-protein repression complex at the different promoters. It has been shown recently that when CytR and CRP are used together to select DNA sequences that are preferred for formation of the three-protein complex, the sequences most commonly selected are CytR recognition motifs separated by 10–13 base pairs and almost centered between CRP1 and CRP2 (8Pedersen H. Valentin-Hansen P. EMBO J. 1997; 16: 2108-2118Crossref PubMed Scopus (47) Google Scholar). This result is surprising because it does not match what is found in the natural promoters, in which CytO is usually located significantly off-center, adjacent to either CRP2 (as indeoP2) or CRP1 (as in cdd, nupG, and udp) with a 2–5-bp spacing between recognition motifs. Nevertheless, a structural model has been proposed in which the DNA is wrapped smoothly around the three-protein complex, and the two CytR DNA binding domains (one per subunit) bind to DNA sequences that are arranged centrosymmetrically and are separated by 11 base pairs (13Kallipolitis B.H. Norregaard M.M. Valentin-Hansen P. Cell. 1997; 89: 1101-1109Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). For a centrosymmetric three-protein complex to form as envisioned in the model, CytR would have to dissociate from CytO and instead bind more widely spaced and symmetrically arranged DNA sequences. Alternatively, CytR, CRP, and/or the DNA would have to be distorted from the symmetric arrangement proposed in the model to accommodate off-center binding by CytR to CytO. Either of these situations would necessarily contribute unfavorably to the stability of the three-protein complex, the former as a result of a decrease in CytR-DNA binding affinity, and the latter as a result of an unfavorable conformational change. Which of these two possible accommodations a particular promoter uses may depend on what alternative, relatively high affinity, CytR-DNA binding sites are provided by the local array of CytR recognition motifs. In these ways, site-specific CytR binding, cooperativity, and competition are inextricably linked. Because cytidine is an effector of CytR-CRP cooperativity, its effect should also be linked to how CytR binds at the various promoters. This linkage might underlie the observed differences between the promoters in effectiveness of induction. To test these hypotheses and to assess how linkage might be involved in differential regulation of transcription, we have investigated cooperative and competitive CytR and CRP binding to the udppromoter. We compare these interactions to those that we investigated previously at the deoP2 promoter. We chose udpfor comparison to deoP2 because these promoters differ substantially in regulatory properties. The ranges of regulated rates of transcription in vivo are about 30-fold for theudp promoter (3Barbier C.S. Short S.A. Senear D.F. J. Biol. Chem. 1997; 272: 16962-16971Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) versus only about 5- to 6-fold for the deoP2 promoter (2Martinussen J. Mollegaard N.E. Holst B. Douthwaite S.R. Valentin-Hansen P. Gralla J.D. DNA-Protein Interactions in Transcription. Alan R. Liss, New York1989: 31-41Google Scholar). CRP is a more effective activator of udp than of deoP2, and CytR is a more effective repressor of udp than of deoP2. Whereas the two promoters do not differ significantly in the arrangement of CRP sites, they do differ in the arrangement of putative CytR binding sites (Fig. 1). Most prominently, the udppromoter contains no putative CytR binding site that occludes CRP1. The results we present here demonstrate that CytR binds specifically to multiple sites at the udp promoter. The results also confirm the expectation that CytR and CRP compete for binding to CRP2 but not for binding to CRP1. However, the most interesting result we found is that cytidine binding to CytR has very different effects on the pattern of CytR-CRP cooperativity at udp from those observed by us at deoP2. Whereas cytidine binding essentially eliminates all CytR-CRP cooperativity in binding to deoP2, it has a very selective effect on CytR-CRP cooperativity in binding toudp. Cytidine binding to CytR largely eliminates pairwise cooperativity between CytR and CRP bound to CRP1 of udp. In contrast, it has a negligible effect on pairwise cooperativity between CytR and CRP bound to CRP2. The net effect on cooperativity in the CRP-CytR-CRP complex is moderate. These results indicate that CytR is highly adaptable to different arrangements of CytR and CRP sites in the absence of cytidine in order to form the three-protein repression complex. However, much of this adaptability appears to be lost when CytR binds cytidine. Thus, the arrangement of the operators might have a substantial influence on induction. An interesting explanation for this behavior can be found in what is known about the structure of CytR and the allosteric mechanism of induction. Crystalline cAMP (>99% pure as free base) was purchased from Sigma. Crystalline cytidine (>99% pure as free acid) was purchased from ICN. Stock concentrations in 50 mm bis-Tris, pH 7.0, and 1 mm EDTA were determined, and purity was assessed spectrophotometrically as described previously (5Perini L.T. Doherty E.A. Werner E. Senear D.F. J. Biol. Chem. 1996; 271: 33242-33255Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). DNase I (code D from Worthington) was treated as described previously (14Brenowitz M. Senear D.F. Shea M.A. Ackers G.K. Methods Enzymol. 1986; 130: 132-181Crossref PubMed Scopus (368) Google Scholar, 15Brenowitz M. Senear D.F. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 2, Supplement 7. Greene Publishing Associates and Wiley-Interscience Associates, New York1989: 12.4.1-12.4.16Google Scholar). [α-32P]deoxynucleotide triphosphates (3,000 Ci/mmol) were purchased from NEN Life Science Products; unlabeled deoxynucleotide triphosphates were obtained from Life Technologies, Inc. Buffer components and reagents were electrophoresis grade, if available; otherwise, they were reagent grade. The CRP preparation used has been described previously (5Perini L.T. Doherty E.A. Werner E. Senear D.F. J. Biol. Chem. 1996; 271: 33242-33255Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). CRP overexpressed from plasmid pPLcCRP1 (16Gronenborn A.M. Clore G.M. Biochem. J. 1986; 236: 643-649Crossref PubMed Scopus (11) Google Scholar) in E. coli strain K12 was isolated to at least 98% purity. The CRP concentration was estimated based on an extinction coefficient of 18,400 m−1 at 280 nm, calculated from the average extinction coefficients for amino acid residues in a protein (17Waxman E. Rusinova E. Hasselbacher C.A. Schwartz G.P. Laws W.R. Ross J.B.A. Anal. Biochem. 1993; 210: 425-428Crossref PubMed Scopus (36) Google Scholar, 18Wetlaufer D.B. Adv. Protein Chem. 1962; 17: 303-390Crossref Scopus (798) Google Scholar). This calculated value is about 10% less than one reported by Takahashi et al. (Ref. 19Takahashi M. Blazy B. Baudras A. Hillen W. J. Mol. Biol. 1989; 207: 783-796Crossref PubMed Scopus (65) Google Scholar; ε(1%) = 9.2 at λmax = 278 nm). CytR was expressed and purified as described previously (3Barbier C.S. Short S.A. Senear D.F. J. Biol. Chem. 1997; 272: 16962-16971Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Protein prepared in this manner is at least 95% CytR dimer under native conditions. However, analysis under denaturing conditions on SDS-polyacrylamide gels indicates variable proteolysis of the CytR by endogenous proteases (5Perini L.T. Doherty E.A. Werner E. Senear D.F. J. Biol. Chem. 1996; 271: 33242-33255Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), similar to what has been observed for other members of the LacI repressor family (20Choi K.Y. Zalkin H. J. Bacteriol. 1992; 174: 6207-6214Crossref PubMed Google Scholar, 21Files J.G. Weber K. J. Biol. Chem. 1976; 251: 3386-3391Abstract Full Text PDF PubMed Google Scholar). The preparations used in these studies contained 70–90% full-length CytR (M r = 37,800). The CytR concentration was estimated using a calculated extinction coefficient of 11,300 ± 800 m−1 at 280 nm (3Barbier C.S. Short S.A. Senear D.F. J. Biol. Chem. 1997; 272: 16962-16971Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The udp DNA fragments used were obtained from plasmid pCB039 (22Barbier C.S. Short S.A. J. Bacteriol. 1992; 174: 2881-2890Crossref PubMed Google Scholar). This plasmid has the regulatory sequence of udp starting 122 base pairs upstream from the transcription start site and through the first the 50 codons of udp (311 bp) cloned into the PstI andEcoRI sites of pUC18. A 498-bp fragment containing thisudp sequence was generated by restriction withPvuII and HindIII. Mutant promoters were generated in which site-specific CRP binding to either CRP1 (CRP1−) or CRP2 (CRP2−) was eliminated by making two bp substitutions in the mutated site. Site-directed mutagenesis was conducted on pCB039 using the QuikChange™ kit from Stratagene. Mutagenic oligonucleotides, 30 nucleotides in length, produced symmetric transitions of G (indicted by underline) to A in both TGTGA CRP recognition motifs of the mutated site. Sequences of the mutants (shown in Fig. 1) were confirmed by dideoxy DNA sequencing. All DNA fragments were purified by agarose gel electrophoresis after banding the plasmid preparations twice in CsCl gradients. DNA was protein-free, as determined from its UV spectrum (23Maniatas T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Fragments were labeled at their HindIII sites using the Klenow fill-in reaction as described previously (15Brenowitz M. Senear D.F. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 2, Supplement 7. Greene Publishing Associates and Wiley-Interscience Associates, New York1989: 12.4.1-12.4.16Google Scholar). Quantitative DNase I footprint titrations were conducted as described previously (5Perini L.T. Doherty E.A. Werner E. Senear D.F. J. Biol. Chem. 1996; 271: 33242-33255Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) in a binding buffer composed of 10 mm bis-Tris (pH 7.00 ± 0.01), 100 mm NaCl, 0.5 mm MgCl2, 0.5 mm CaCl2, 50 μg/ml bovine serum albumin, and 1 μg/ml calf thymus DNA. Experiments in which CRP was present also contained 150 μm cAMP. This cAMP concentration maximizes the fraction of CRP present as the active DNA-binding species in which cAMP is bound to one but not both of the subunits (denoted by CRP(cAMP)1). Under these conditions, the active fraction is 0.64 ± 0.02 (5Perini L.T. Doherty E.A. Werner E. Senear D.F. J. Biol. Chem. 1996; 271: 33242-33255Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Binding reaction mixtures (200 μl) were exposed to 2–6 ng of DNase I added in a 5.0 μl volume for 12.0 min and quenched by the addition of 0.2 volume of 50 mm Na4EDTA before the addition of stop solution (24Senear D.F. Batey R. Biochemistry. 1991; 30: 6677-6688Crossref PubMed Scopus (55) Google Scholar). Dried gels were imaged using a Molecular Dynamics PhosphorImager 435SI. Phosphor plates were exposed for 3–4 days and scanned at 176 μm spatial resolution. Analysis of the digital images was conducted using the ImageQuant program (Molecular Dynamics) essentially as described previously (15Brenowitz M. Senear D.F. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 2, Supplement 7. Greene Publishing Associates and Wiley-Interscience Associates, New York1989: 12.4.1-12.4.16Google Scholar, 24Senear D.F. Batey R. Biochemistry. 1991; 30: 6677-6688Crossref PubMed Scopus (55) Google Scholar). The individual site binding data were first analyzed separately for each site to obtain the Gibbs free energy change corresponding tok (ΔG i = −RTln k) in Equation 1. Yi=Po+(Pmax−Po)·k·L1+k·LEquation 1 In Equation 1, Y i is the fractional saturation of binding site i at the free protein ligand concentration L, k is the association constant, and P o andP max are the baseline and maximum fractional protection for a given titration (14Brenowitz M. Senear D.F. Shea M.A. Ackers G.K. Methods Enzymol. 1986; 130: 132-181Crossref PubMed Scopus (368) Google Scholar). For simple binding of either CRP or CytR alone, Equation 1 gives the intrinsic free energy change for local binding, ΔG i. For binding experiments in which both CytR and CRP are present, analysis according to Equation 1provides an accurate estimate of the individual site loading free energy change, ΔG l,i (25Ackers G.K. Shea M.A. Smith F.R. J. Mol. Biol. 1983; 170: 223-242Crossref PubMed Scopus (110) Google Scholar), and its confidence limits (26Senear D.F. Ackers G.K. Biochemistry. 1990; 29: 6568-6577Crossref PubMed Scopus (45) Google Scholar, 27Senear D.F. Perini L.T. Gavigan S.A. Methods Enzymol. 1998; 295: 403-424Crossref PubMed Scopus (6) Google Scholar). Subsequently, global analysis of the individual site CRP and CytR binding data was conducted using equations that describe cooperative and competitive binding of CRP and CytR according to the model defined by the promoter configurations specified in Table II. Equations to describe the binding to each of the individual sites were derived by considering the relative probability of each promoter configuration as given by the following equation. fs=e−ΔGs/RT⋅[CRP(cAMP)1]i⋅[CytR]j∑sije−ΔGs/RT⋅[CRP(cAMP)1]i⋅[CytR]jEquation 2 ΔG is the sum of free energy contributions for configuration s (Table III); i and jare the stoichiometries of bound CRP(cAMP)1 complexes and CytR dimers in configuration s. Summation of the relative probabilities for all configurations in which protein is bound to any given site derives the binding equation for that site. For reduced valence, operators CRP1− and CRP2−, configurations in which CRP(cAMP)1 is bound to the mutated site were excluded from the summation.Table IIConfigurations and free energy states for CRP and CytR binding to the udp promoterOperator configurationFree energy contributionsFree energy stateCRP2CytR at CRP2CytRCRP11OOOOReference stateΔG s,12OOOCRPΔG 1ΔG s,23CRPOOOΔG 2ΔG s,34OOCytROΔG 3ΔG s,45OCytROOΔG 4ΔG s,56CRPOOCRPΔG 1 + ΔG 2ΔG s,67OOCytRCRPΔG 1 + ΔG 3 + ΔG 13ΔG s,78OCytROCRPΔG 1 + ΔG 4ΔG s,89CRPOCytROΔG 2 + ΔG 3 + ΔG 23ΔG s,910OCytRCytROΔG 3 + ΔG 4ΔG s,1011CRPOCtyRCRPΔG 1 + ΔG 2 + ΔG 3 + ΔG 123ΔG s,1112OCytRCytRCRPΔG 1 + ΔG 3 + ΔG 4 + ΔG 13ΔG s,12udp promoter configurations with sites denoted as filled (CRP or CytR) or empty (O). CytR binding to the site(s) that overlaps and occludes CRP2 is denoted by [CytR]. The total Gibbs free energy of each configuration relative to the unliganded reference state is given as a sum of contributions from four free energy changes for intrinsic binding of CRP and CytR (ΔG k) and three free energy changes for cooperative interaction between liganded sites (ΔG ij(k)). Subscripts denote the liganded sites: 1, CRP1; 2, CRP2; 3, intervening CytR site; 4, CytR site(s) that overlaps and occludes CRP2. Open table in a new tab Table ILoading free energy changes for binding of CRP and CytR to the udp regulatory regionudpvalenceTitrantEffectorsaEffector concentrations: CRP, 0.1 μm(total dimer); cAMP, 150 μm (present in all experiments); CytR, 75 nm (dimer); cytidine, 1 mm.No. of experimentsbΔG l values shown are the means of multiple determinations (± S.D.). The number of separate experiments represented in the means is indicated.Operator siteCRP2CytR at CRP2CytRCRP1AllCRPNone4−13.5 ± 0.3—−12.0 ± 0.1AllCytRNone13—−10.7 ± 0.4—WT/CRP1−CytRNone8−9.9 ± 0.5—CRP2−CytRNone3−10.7 ± 0.2—Wild typeCRPCytR2−13.9 ± 0.1—−13.5 ± 0.1CRP1−CRPCytR2−13.7 ± 0.4——CRP2−CRPCytR2——−12.9 ± 0.1Wild typeCytRCRP5—−12.8 ± 0.2—CRP1−CytRCRP3—−12.4 ± 0.3—CRP2−CytRCRP4—−12.7 ± 0.2—Wild typeCytRCRP, cytidine3—−12.2 ± 0.4—CRP1−CytRCRP, cytidine2—−11.9 ± 0.3—CRP2−CytRCRP, cytidine2—−11.2 ± 0.1—Free energy changes for saturation of udp operators with either CRP(cAMP)1 or CytR in the presence or absence of effector ligands are as indicated. Values of ΔG l(in kcal/mol) were determined by an analysis of individual site binding curves as described in the text.a Effector concentrations: CRP, 0.1 μm(total dimer); cAMP, 150 μm (present in all experiments); CytR, 75 nm (dimer); cytidine, 1 mm.b ΔG l values shown are the means of multiple determinations (± S.D.). The number of separate experiments represented in the means is indicated. Open table in a new tab Table IIIGlobal analysis of individual site binding data from DNase I footprint titrationsParameterValuebΔG values are in kcal/mol ± 65% confidence limits.ΔG 1−12.0 ± 0.2ΔG 2−13.7 ± 0.2ΔG 3−10.9 ± 0.2ΔG 4−10.5 ± 0.4ΔG 13−1.4 ± 0.2ΔG 23−1.3 ± 0.3ΔG 123−2.3 ± 0.3ΔΔG 13+1.0 ± 0.3ΔΔG 23+0.3 ± 0.3ΔΔG 123+0.8 ± 0.4s0.058cSquare root of the variance of the fitted curves.Parameter values were obtained from global analysis of the individual site protection data represented in Table I according to the model formulated in Table II.aCytR binding data for only the previously recognized operator (designa" @default.
• W2007127972 created "2016-06-24" @default.
• W2007127972 creator A5020382119 @default.
• W2007127972 creator A5031220394 @default.
• W2007127972 creator A5046867700 @default.
• W2007127972 creator A5087857623 @default.
• W2007127972 date "1999-06-01" @default.
• W2007127972 modified "2023-10-15" @default.
• W2007127972 title "Role of Multiple CytR Binding Sites on Cooperativity, Competition, and Induction at the Escherichia coli udpPromoter" @default.
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