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• W2075565562 abstract "Transcription from cistrons of theEscherichia 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 Asp281or 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. Transcription from cistrons of theEscherichia 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 Asp281or 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. The transport proteins and enzymes required for nucleoside utilization in Escherichia coli are encoded by genes belonging to the CytR regulon (1Hammer-Jespersen K. Munch-Petersen A. Metabolism of Nucleotides, Nucleosides and Nucleobases in Microorganisms. Academic Press, London1983: 203-258Google Scholar). This gene family consists of nine unlinked transcriptional units whose expression is coordinately controlled by the interplay of two gene regulatory proteins. Transcription is activated in response to intracellular cAMP levels by CRP 1The abbreviations used are: CRP, E. coli cAMP receptor protein (CRP is also referred to as CAP, catabolite activator protein); CID, cytidine induction defective; MWC, Monod Wyman and Changeux; KNF, Koshland Nemethy and Filmer; CDA, cytidine deaminase; UDP, uridine dephosphorylase; Cmr, chloramphenicol resistant; Amps, ampicillin-sensitive; MOPS, 4-morpholinepropanesulfonic acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. 1The abbreviations used are: CRP, E. coli cAMP receptor protein (CRP is also referred to as CAP, catabolite activator protein); CID, cytidine induction defective; MWC, Monod Wyman and Changeux; KNF, Koshland Nemethy and Filmer; CDA, cytidine deaminase; UDP, uridine dephosphorylase; Cmr, chloramphenicol resistant; Amps, ampicillin-sensitive; MOPS, 4-morpholinepropanesulfonic acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. and repressed by a three-protein, CRP· CytR·CRP, complex. Transcription is induced when CytR binds cytidine. A central feature of this coordinate regulation is that CytR and CRP bind cooperatively to their respective operators (2Pedersen H. Sogaard-Andersen L. Holst B. Valentin-Hansen P. J. Biol. Chem. 1991; 266: 17804-17808Abstract Full Text PDF PubMed Google Scholar). This is so despite the role of CytR as a functional antagonist of CRP. The critical role that cooperativity plays is highlighted by the fact that expression is induced, because this cooperative interaction is lost when CytR binds cytidine. Cytidine binding has no effect on intrinsic CytR binding to DNA. CytR is a member of the LacI family of bacterial repressors (3Weickert M.J. Adhya S. J. Biol. Chem. 1992; 267: 15869-15874Abstract Full Text PDF PubMed Google Scholar). The gene regulatory activity of each of these proteins is modulated by binding a peripheral ligand, which functions as either inducer or co-repressor. 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 co-repressor 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 (4Schumacher M.A. Choi K.Y. Lu F. Zalkin H. Brennan R.G. Cell. 1995; 83: 147-155Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 5Schumacher M.A. Choi K.Y. Zalkin H. Brennan R.G. Science. 1994; 266: 763-770Crossref PubMed Scopus (335) Google Scholar, 6Lewis M. Chang G. Horton N.C. Kercher M.A. Pace H.C. Schumacher M.A. Brennan R.G. Lu P. Science. 1996; 271: 1247-1254Crossref PubMed Scopus (655) Google Scholar). In these two cases, binding of co-repressor or inducer, respectively, causes a change in tertiary structure that alters substantially the dimer interface. In the non-DNA binding conformation, hinge helices that connect the helix-turn-helix motif to the ligand binding globular core domain are destabilized, and the helix-turn-helix motifs from the two subunits are thought to be out of register with successive DNA major grooves. In this manner, cooperative ligand binding (7Daly T.J. Matthews K.S. Biochemistry. 1986; 25: 5479-5484Crossref PubMed Scopus (27) Google Scholar, 8Choi K.Y. Zalkin H. J. Bacteriol. 1992; 174: 6207-6214Crossref PubMed Google Scholar) to the individual subunits controls a concerted quarternary conformational change of the dimer. These features are consistent with MWC allostery. While the structural mechanisms that couple ligand binding to tertiary conformation differ in the two proteins (4Schumacher M.A. Choi K.Y. Lu F. Zalkin H. Brennan R.G. Cell. 1995; 83: 147-155Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 5Schumacher M.A. Choi K.Y. Zalkin H. Brennan R.G. Science. 1994; 266: 763-770Crossref PubMed Scopus (335) Google Scholar, 6Lewis M. Chang G. Horton N.C. Kercher M.A. Pace H.C. Schumacher M.A. Brennan R.G. Lu P. Science. 1996; 271: 1247-1254Crossref PubMed Scopus (655) Google Scholar), the tertiary and quarternary structural perturbations are remarkably similar. The structures of the LacI family proteins, including CytR, appear to be highly conserved (5Schumacher M.A. Choi K.Y. Zalkin H. Brennan R.G. Science. 1994; 266: 763-770Crossref PubMed Scopus (335) Google Scholar, 6Lewis M. Chang G. Horton N.C. Kercher M.A. Pace H.C. Schumacher M.A. Brennan R.G. Lu P. Science. 1996; 271: 1247-1254Crossref PubMed Scopus (655) Google Scholar, 9Hsieh M. Hensley P. Brenowitz M. Fetrow J.S. J. Biol. Chem. 1994; 269: 13825-13835Abstract Full Text PDF PubMed Google Scholar, 10Schumacher M.A. Macdonald J.R. Bjorkman J. Mowbray S.L. Brennan R.G. J. Biol. Chem. 1993; 268: 12282-12288Abstract Full Text PDF PubMed Google Scholar). Given the structural resemblance among family members plus the similarity of allosteric mechanism for LacI and PurR, a similar mechanism might be anticipated for CytR. Yet CytR differs from all LacI family members in that it is cooperativity that is allosterically controlled and not intrinsic DNA binding. Allostery thus appears to have a different structural basis in CytR than in other LacI/PurR proteins. Understanding the allosteric mechanism is central to understanding coordinate regulation of the CytR regulon genes. Recently, we showed that CytR binds to multiple operators at one CytR regulated promoter,deoP2 (12Perini 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 binding to the operator responsible for repression interacts cooperatively with CRP binding to flanking CRP sites, CRP1 and CRP2. However, by binding to additional specific sites, CytR competes with CRP for binding to CRP1 and CRP2. The net result of cooperativity and competition is that while CRP recruits CytR to form the repression complex, there is no significant reciprocal recruitment of CRP by CytR. This effect has also been reported for thenupG promoter (13Pedersen H. Dall J. Dandanell G. Valentin-Hansen P. Mol. Microbiol. 1995; 17: 843-853Crossref PubMed Scopus (24) Google Scholar). These interactions presumably function to direct both a multistage activation of transcription, using both Class I and Class II CRP mechanisms (14Ebright R.H. Mol. Microbiol. 1993; 8: 797-802Crossref PubMed Scopus (167) Google Scholar) and also a similar multistage repression mediated by CytR. We have proposed that this might be a general feature of CytR-mediated gene regulation (12Perini 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). The unique mechanism of cytidine mediated induction also suggests a multistage process. The cooperativity to which cytidine binding is linked appears to be complementary pair wise in nature. This follows from the observation that the free energy change characterizing cooperativity in the three protein complex, CRP·CytR·CRP bound to DNA, is equal to the sum of free energy changes characterizing pair wise cooperativity between CytR and CRP bound either to CRP1 or to CRP2 (12Perini 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). If cooperativity in the three-protein repression complex is pairwise, then it is easy to envision that the two subunits of the dimer might react independently to cytidine binding. This would result in sequential elimination of pairwise, CytR·CRP cooperativity, hence sequential relief from repression, in response to sequential cytidine binding to the subunits. The most general possibilities for coupling between ligand binding and transcription initiation are presented in Fig. 1. We have combined biophysical chemical and molecular genetic approaches to investigate these possibilities. First, CytR binding to CRP-saturateddeoP2 was analyzed to evaluate the total contribution from cooperativity. Subsequently, CytR binding titrations were conducted as a function of cytidine concentration. The shape of the transition characterizing loss of cooperativity as cytidine binds CytR indicates that induction is an all or nothing process that occurs concomitant with only one of the cytidine binding steps. Second, CytR mutants were isolated and characterized as fully functional repressors, but which do not induce. The only defect in these mutants is inability to bind cytidine. By co-expressing cytidine induction-defective subunits and wild-type subunits, we evaluated whether the resulting heterodimers would support induction with only one subunit capable of binding cytidine. The combined data from these studies indicate that induction results when cytidine binds to the first subunit of the CytR dimer.Figure 1Schematic of CytR cooperativity states reflecting different allosteric models. CRP and CytR are shown bound to their respective operators, CRP1, CRP2, and CytO.Shading and object shape are used to distinguish differences in tertiary and quarternary states, respectively. Cooperativity is indicated by contact between adjacent proteins. Only the favored state is shown in each case, and symmetrically redundant configurations are not shown. A represents a concerted model featuring an equilibrium between pre-existing states. Cytidine binding shifts the poise of the equilibrium from favoring the cooperative state to favoring the noncooperative state. This is MWC allostery.B represents a sequential allosteric model in which cytidine binding induces a conformational change of only the subunit to which it binds. This eliminates cooperative interaction between that subunit and the adjacent DNA-bound CRP·cAMP and also affects interactions with the remaining subunit to affect its affinity for cytidine binding. This is KNF allostery. C represents sequential but concerted transitions between cooperativity states. Unliganded CytR interacts cooperatively with CRP·cAMP complexes bound to both flanking sites, CRP1 and CRP2. Binding of cytidine to the first subunit induces a state in which neither subunit interacts with adjacent bound CRP·cAMP. A similar model in which the obligatory transition to noncooperative binding occurs only when the second cytidine binds is also possible.View Large Image Figure ViewerDownload Hi-res image Download (PPT) TableI lists the bacterial strains and plasmids used in this study. The CID cytR allele, cytRD281N, was transferred to the bacterial chromosome as described by Winans et al. (15Winans S.C. Elledge S.J. Krueger J.H. Walker G.C. J. Bacteriol. 1985; 161: 1219-1221Crossref PubMed Google Scholar). First, the cat gene was inserted into plasmid pCB071-161 at a position 44 bp 3′ of the cytR termination codon, resulting in plasmid, pCB122. Second, E. coli strain VJS803 was transformed with linearized pCB122, and a recombinant strain, SS6140, was selected as chloramphenicol-resistant (Cmr) and ampicillin-sensitive (Amps). ThecytRD281N allele was subsequently transferred to other strains by P1 transduction. The presence of the cytRD281Nallele was verified in each Cmr isolate by enzyme assays. The tsx-lac gene fusions carried by strains GP4, Tsx-lac500, Tsx-lac501, Tsx-lac502 and Tsx-lac503 (16Gerlach P. Soogard-Andersen L. Pedersen H. Martinussen J. Valentin-Hansen P. J. Bacteriol. 1991; 173: 5419-5430Crossref PubMed Scopus (36) Google Scholar) were transferred into strain SS6003 by P1 transduction. The cytR::Tn10dTet insertion was then moved from SS6018 into each SS6003 derivative, yielding strains SS6117 through SS6121 (Table I).Table IBacterial strains, plasmids, and bacteriophages used in these studiesCharacteristicsSourceE. coli strains SS6003F− thi leu rspL Δ(argF-lac) U169S. Short SS6004SS6003, Φ(udp-lac)6 (Hyb) (λRS45)S. Short SS6018SS6003, Φ(udp-lac)8(λRS45) cytR∷Tn10 dTetS. Short SS6083SS6004, recA56srlC300∷Tn10S. Short SS6117SS6003, Φ(tsx-lac)500 (λplac Mu55)cytR∷Tn10 dTetS. Short SS6118SS6003, Φ(tsx-lac)501 (λplac Mu55)cytR∷Tn10 dTetS. Short SS6119SS6003, Φ(tsx-lac)502 (λplac Mu55)cytR∷Tn10 dTetS. Short SS6120SS6003, Φ(tsx-lac)503 (λplac Mu55)cytR∷Tn10 dTetS. Short SS6121SS6003, Φ(tsx-lac) (λplac Mu55)cytR∷Tn10 dTetS. Short SS6140VJS803, cytR D281NThis study SS6141SS6004, cytR D281NThis studyBL21(DE3)F− hsdS gal λDE3F. W. Studier JM103Δ(lac-pro) thi rpsL supE endA sbcB15/F′traD36 proAB lacI q lacZΔM15J. MessingMC4100F− araD139 Δ(arg-lac)U169 rpsL150 relA1 flb5301 deoC1 ptsF25 rbsRM. CasadabanVJS803recB21 recC22 sbcB15 sbcC201 argE3 his-4 leuB6 proA2 thr-1 ara-14 galK2 Δ(arg-lac)U169 Δ(trpEA)2 mtl-1 xyl-5 thi-1 rpsL31 supE44 tsx-33V. StewartPlasmids pCB071–161AmpR, an NdeI−, Cla I−,1-aThe superscript − indicates that the designated endonuclease cleavage site has been removed by reaction with either the Klenow fragment of DNA polymerase I or with T4 DNA polymerase. pBR322 derivative carrying cid allele cytRD281NS. Short pCB093A KanR pCB071 derivative carrying the wild-typecytR geneS. Short pCB094A KanR pCB093 derivative lacking the cytR gene and flanking sequenceS. Short pCB095A KanR pGLP4 derivative carrying the wild-type cytR gene and flanking sequenceS. Short pCB096A KanR pCB093 derivative deleted only for thecytR coding sequenceS. Short pCB122An AmpR pCB071–161 derivative. The cat gene from pACYC184 has been inserted into the AflII site 44 bp 3′ from the cytR termination codonThis study pCB123A KanR pSS584 derivative carrying the wild-type cytRgeneThis study pCB124A KanR pSS584 derivative carrying CytRD281NThis study pCB127A KanR pSS584 derivative carryingCytRM151VThis study pCB128A KanRpSS584 derivative carrying CytRM151IThis study pCB131A KanR pSS584 derivative carryingCytRV15AThis study pGLP4A KanRpACYC184 derivativeS. Short pSS584A KanR pBR322 derivative. Contains the galK coding sequence fused to a T7 promoterS. Short pSS1332An AmpR pUC19 derivative with an 858-bp insert containing the deoPIP2 region of thedeo operonS. ShortBacteriophages M13mp19-cytR10M13mp19 derivative containing thecytR coding sequence bounded by SmaI andBamHIThis study1-a The superscript − indicates that the designated endonuclease cleavage site has been removed by reaction with either the Klenow fragment of DNA polymerase I or with T4 DNA polymerase. Open table in a new tab To express CytR, the coding sequences of wild-type and mutantcytR alleles were subcloned as anNdeI/BamHI fragment downstream of the T7 promoter carried by plasmid pSS584. Strain BL21(DE3) (17Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4811) Google Scholar, 18Rosenberg A.H. Lade B.N. Chui D.S. Lin S.W. Dunn J.J. Studier F.W. Gene ( Amst .). 1987; 56: 125-135Crossref PubMed Scopus (1043) Google Scholar) was transformed with each construct. The control plasmid for these experiments is pCB135, a pSS584 derivative devoid of cytR coding sequence. Bacteria were collected from exponentially growing cultures for enzyme assays. The medium contained Vogel and Bonner salts (19Vogel H.J. Bonner D.M. J. Biol. Chem. 1956; 218: 97-106Abstract Full Text PDF PubMed Google Scholar) supplemented with vitamin B1 at 5 μg/ml, 0.02% casamino acids, and 0.4% glycerol (20Barbier C.S. Short S.A. J. Bacteriol. 1992; 174: 2881-2890Crossref PubMed Google Scholar). BL21(DE3) derivatives harboring CytR plasmids were grown in a 1% Bacto-tryptone, 0.4% glycerol medium containing Vogel and Bonner salts (TV medium). Either L-broth or 2 × YT was used for transformations and plasmid preparations (21Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). The Lac ± phenotypes of the various strains were determined on solid TTC-Lac medium as described previously (20Barbier C.S. Short S.A. J. Bacteriol. 1992; 174: 2881-2890Crossref PubMed Google Scholar). When used, the final cytidine concentration was 2 mm. Antibiotic concentrations used in the media were: ampicillin, 100 μg/ml; tetracycline, 15 μg/ml; chloramphenicol, 20 μg/ml; and kanamycin, 25 μg/ml in minimal medium or 50 μg/ml in rich medium. A mixture of mutagenic oligonucleotides complementary tocytR codons 276 through 284 was synthesized using an Applied Biosystems model 381A DNA synthesizer. The spiking protocol of Hutchison et al. (22Hutchison III, C.A. Nordeen S.K. Vogt K. Edgell M.H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 710-714Crossref PubMed Scopus (73) Google Scholar, 23Hutchison III, C.A. Swanstrom R. Loeb D.D. Methods Enzymol. 1991; 202: 356-390Crossref PubMed Scopus (20) Google Scholar) was used to create degeneracy in the oligonucleotide sequence. The mutagenic oligonucleotide mixture and a site-directed mutagenesis kit from Amersham Corp. was used to mutate the cytR gene on an M13mp19cytR10 template. Both single and multiple mutations were obtained, the frequency of single mutations being about 30%. Phage pooled from about 5000 mutagenized M13mp19cytR10 plaques was propagated in E. coli strain JM103 by incubation for 4 h in 2 × YT medium. RF-M13 DNA was prepared as described (24Maniatas T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 4.29-4.32Google Scholar). The cytR gene fragment containing the mutagenized sequence bounded by ApaI andBamHI cleavage sites was subcloned into pCB093 by fragment exchange (20Barbier C.S. Short S.A. J. Bacteriol. 1992; 174: 2881-2890Crossref PubMed Google Scholar). The recombinant plasmid pool was transferred into SS6018 (cytR), which was grown on TTC-Lac-Kan medium containing 2 mm cytidine, to identify mutant CID repressors. The dominant negative phenotype of CytR mutants was established using CytR+ strain, SS6004, as described previously (20Barbier C.S. Short S.A. J. Bacteriol. 1992; 174: 2881-2890Crossref PubMed Google Scholar). The steady-state level of wild-type and mutant CytR was measured using a Western immunoblot analysis (20Barbier C.S. Short S.A. J. Bacteriol. 1992; 174: 2881-2890Crossref PubMed Google Scholar). Each cytR mutation that yielded a stable mutant protein was identified by DNA sequencing of the mutagenized cytR gene segment on a purified, double-stranded template (20Barbier C.S. Short S.A. J. Bacteriol. 1992; 174: 2881-2890Crossref PubMed Google Scholar). Bacteria used for enzyme assays were grown and cell extracts prepared as described previously (25Singer J.T. Barbier C.S. Short S.A. J. Bacteriol. 1985; 163: 1095-1100Crossref PubMed Google Scholar). Cytidine deaminase (CDA) and uridine dephosphorylase (UDP) spectroscopic assays were performed as described previously (25Singer J.T. Barbier C.S. Short S.A. J. Bacteriol. 1985; 163: 1095-1100Crossref PubMed Google Scholar, 26Short S.A. Singer J.T. Gene ( Amst .). 1984; 31: 205-211Crossref PubMed Scopus (9) Google Scholar) except that the CDA assay mixture contained 50 mm Tris-HCl (pH 7.5) and 0.5 mm cytidine. The β-galactosidase activity of exponentially growing bacteria was measured as described by Miller (21Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). To express the enzyme activity as specific activity of the cell extracts, the total protein concentration of the extracts was measured by the Bradford assay (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar) using bovine serum albumin as a standard. BL21(DE3) derivatives harboring T7 expression plasmids for either wild-type or mutant CytR were grown at 37 °C in nucleoside-free TV medium with 0.4% glycerol toA 600 ≈ 1.0. CytR expression was induced by adding 1% lactose and 2 mmisopropyl-β-d-thiogalactopyranoside and incubating for 60 min before harvesting the cells. Cell pellets were washed, resuspended in 20 mm MOPS (pH 6.8), 2 mm MgSO4, 1 mm Na4EDTA, 200 mm NaCl, and treated with lysozyme, added to 10 μg/ml. The cells were frozen at −20 °C, thawed at 23 °C, and broken by sonication. The final cell extract was the clear supernatant remaining following centrifugation at 100,000 × g for 1 h at 4 °C. CytR was purified using a simpler protocol than that reported several years ago (2Pedersen H. Sogaard-Andersen L. Holst B. Valentin-Hansen P. J. Biol. Chem. 1991; 266: 17804-17808Abstract Full Text PDF PubMed Google Scholar) but which yielded a higher yield of CytR with similar purity. All purification steps were carried out at 4 °C. Pellets from cells harvested 165 min postinduction were resuspended in 20 mm MOPS (pH 6.80), 2 mm MgSO4, 1 mm Na4EDTA, 1 mm dithiothreitol (R-buffer) supplemented with 0.3m NaCl. The resuspended cells were lysed using two passes through a French press and centrifuged at 50,000 × gfor 3 h. The supernatant was adjusted to 0.2 m NaCl and 10% glycerol in R-buffer. Polyethyleneimine was added to a final concentration of 0.04% to precipitate nucleic acids and some proteins. The supernatant from a low speed centrifugation was adjusted to 0.1m NaCl and chromatographed on two Bio-Rad EconoPac Q cartridges (5 ml each) connected in series using a Pharmacia FPLC. The pooled CytR containing the flow-through peak was loaded on two Bio-Rad Econo-Pac S cartridges connected in series. After washing the column with 0.2 m NaCl R-buffer until theA 280 of the wash returned to the buffer base line, the column was eluted using a 0.2–0.6 m NaCl gradient. CytR elutes in a broad peak between 0.3 and 0.4 mNaCl. CytR concentration was estimated from an extinction coefficient of 0.30 ± 0.03 cm−1 mg−1 ml at 280 nm. This value was calculated from the average extinction coefficients for amino acid residues in a protein (28Waxman E. Rusinova E. Hasselbacher C.A. Schwartz G.P. Laws W.R. Ross J.B. Anal. Biochem. 1993; 210: 425-428Crossref PubMed Scopus (36) Google Scholar, 29Wetlaufer D.B. Adv. Protein Chem. 1962; 17: 303-390Crossref Scopus (789) Google Scholar, 30Perkins S.J. Sim R.B. Eur. J. Biochem. 1986; 157: 155-168Crossref PubMed Scopus (46) Google Scholar, 31Mach H. Middaugh C.R. Lewis R.V. Anal. Biochem. 1992; 200: 74-80Crossref PubMed Scopus (425) Google Scholar). The unusually low extinction is due to the fact that CytR contains no tryptophan. Based on the calculated extinction, the yield of CytR using this expression and purification protocol is 1.5–2 mg/g of cell paste. CytR is stored at a concentration of 2–3 mg/ml in the S-column elution buffer in a liquid nitrogen dewer after flash freezing as ∼25-μl beads in liquid nitrogen. The DNA and cytidine binding activities remain stable for at least several years when stored in this manner. Sedimentation equilibrium analysis shows this material to be homogeneous dimer, with no evidence for either dissociation to monomer or association to higher order polymers over the concentration range, 0.1–10 μm. 2J. Wool, D. F. Senear, and T. M. Laue, unpublished observations. More recent analysis of gel mobility shift assays of CytR binding to DNA has been interpreted to indicate that CytR remains as stable dimer over the range of concentrations at which it binds DNA operators (11Kristensen H.H. Valentin-Hansen P. Sogaard-Andersen L. J. Mol. Biol. 1996; 260(2): 113-119Crossref Scopus (19) Google Scholar). Based on these data, the binding experiments were analyzed according to the simplest model in which CytR exists only as dimer. Binding of [3H]cytidine to purified wild-type CytR and to both wild-type and mutant CytR containing cell-free extracts was measured using a filter binding assay. Binding reaction mixtures contained either 18–50 nm purified CytR dimer or 15–30 μg of cell extract protein in a 100-μl volume containing 0.04–11.0 μm [3H]cytidine (NEN Life Science Products). Two different buffers were used: 1) 20 mm MOPS (pH 6.8), 2 mm MgSO4, 1 mm NaEDTA, 200 mm NaCl and 2) 10 mm bis-Tris (pH 7.0), 100 mm NaCl, 0.5 mm MgCl2, 0.5 mm CaCl2. Both buffers contained 100 μg/ml bovine serum albumin and 1 μg/ml calf thymus DNA. Following a 5-min incubation at 23 °C, the CytR-bound [3H]cytidine contained in 80 μl of assay mix was collected on a prewashed nitrocellulose filter (Millipore HAWP 02500; Millipore Corp., Bedford, MA). The filters were washed once with 500 μl of assay buffer, air-dried, and then dissolved in 3.5 ml of Packard Filter-Count LSC mixture (Packard Instrument Co.). Radioactivity was measured using a Packard model 1900TR scintillation counter. For determination of nucleoside binding constants, binding assays were conducted as titrations by varying the nucleoside concentration at constant CytR concentration. The data were analyzed according to a simple Langmuir binding model as described below (see Equation 1) using a nonlinear least squares curve fitting program (32Leatherbarrow, R. J. (1990) GraFit Version 2.0, Erithacus Software Ltd., Staines, UK.Google Scholar). The CytR concentration used in some titrations was not negligible. To analyze data under these conditions, the conservation polynomials for total cytidine and total CytR were solved for the concentration of free cytidine for each data point and at each iteration of the nonlinear least squares analysis. The program NONLN (33Johnson M.L. Frasier S.G. Methods Enzymol. 1985; 117: 301-342Crossref Scopus (510) Google Scholar) was used for this purpose. In experiments to compare [3H]cytidine binding by CytR mutants in cell-free extracts and wild-type CytR, the CytR content of the cell-free extracts was estimated using Western immunoblots as described above. For each extract, 25–200 ng of extract protein was electrophoresed on SDS-16.5% acrylamide gels. Proteins were electrotransferred to Immobilon-P membranes (Millipore Corp.) as described previously (20Barbier C.S. Short S.A. J. Bacteriol. 1992; 174: 2881-2890Crossref PubMed Google Scholar). CytR was complexed with anti-CytR antibody and 125I-protein A. 125I-Protein A in the complex was quantitated using a Molecular Dynamics PhosphorIm" @default.
• W2075565562 created "2016-06-24" @default.
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• W2075565562 creator A5046867700 @default.
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• W2075565562 date "1997-07-01" @default.
• W2075565562 modified "2023-10-16" @default.
• W2075565562 title "Allosteric Mechanism of Induction of CytR-regulated Gene Expression" @default.
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