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• W2068372232 abstract "Previous studies demonstrated that bacteriochlorophyll, carotenoid, and light harvesting gene expression in Rhodobacter capsulatus is repressed under aerobic growth conditions by the repressor CrtJ. Isolated CrtJ is known to bind to the palindrome TGTN12ACA, which is present in two copies in thebchC promoter, one of which spans the −35 and the other the −10 ς-70 recognition sequences. In this study, we demonstrate that CrtJ binds to the two palindromic sites in the bchCpromoter in a cooperative manner. The level of cooperativity of CrtJ binding to the −35 palindrome was shown to be 26-fold. A distance of 8 base pairs between the two palindromic sites was shown to be critical for cooperative binding, as evidenced by the disruption of binding that resulted when +6 and +11 base pairs were inserted between the palindromes. Previous studies demonstrated that bacteriochlorophyll, carotenoid, and light harvesting gene expression in Rhodobacter capsulatus is repressed under aerobic growth conditions by the repressor CrtJ. Isolated CrtJ is known to bind to the palindrome TGTN12ACA, which is present in two copies in thebchC promoter, one of which spans the −35 and the other the −10 ς-70 recognition sequences. In this study, we demonstrate that CrtJ binds to the two palindromic sites in the bchCpromoter in a cooperative manner. The level of cooperativity of CrtJ binding to the −35 palindrome was shown to be 26-fold. A distance of 8 base pairs between the two palindromic sites was shown to be critical for cooperative binding, as evidenced by the disruption of binding that resulted when +6 and +11 base pairs were inserted between the palindromes. base pair(s) polymerase chain reaction N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Tris-borate EDTA. The purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus synthesizes a photosystem under anaerobic growth conditions. Regulation of photosystem synthesis is primarily mediated by controlling the initiation of photosynthesis gene expression (reviewed in Refs. 1Bauer C.E. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 1221-1234Google Scholar and 2Bauer C.E. Bird T.H. Cell. 1996; 85: 5-8Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In a previous study, we demonstrated that aerobic repression of bacteriochlorophyll (bch), carotenoid (crt), and light harvesting II (puc) gene expression involves the transcription factor CrtJ (3Ponnampalam S.N. Buggy J.J. Bauer C.E. J. Bacteriol. 1995; 177: 2990-2997Crossref PubMed Google Scholar, 4Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1997; 272: 18391-18396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Biochemical analysis of purified CrtJ indicates that it functions as a redox-sensitive DNA-binding protein that optimally binds to thebchC promoter under oxidizing conditions (4Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1997; 272: 18391-18396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). DNase I footprint titration assays of CrtJ binding to thebchC promoter region indicates that CrtJ protects two conserved palindromes (TGTN12ACA), one of which spans the −35 and the other the −10 ς-70 sequence recognition motifs (3Ponnampalam S.N. Buggy J.J. Bauer C.E. J. Bacteriol. 1995; 177: 2990-2997Crossref PubMed Google Scholar). Hill coefficient determination from DNA binding isotherms indicate that CrtJ binds to the bchC promoter region as a tetramer, presumably with CrtJ monomers interacting with each palindrome half site (4Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1997; 272: 18391-18396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Inspection of promoters that are regulated by CrtJ indicates that there are two motifs (Fig. 1). One class, represented by the bchC promoters of R. capsulatus and Rhodobacter sphaeroides and by theR. sphaeroides puc promoter, contains two CrtJ recognition palindromes, one of which spans the −35 and the other the −10 ς-70 recognition sequences (4Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1997; 272: 18391-18396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 5Ma D. Cook D.N. O'Brien D.A. Hearst J.E. J. Bacteriol. 1993; 175: 2037-2045Crossref PubMed Google Scholar, 6Wellington C.L. Beatty J.T. Gene. 1989; 83: 251-261Crossref PubMed Scopus (13) Google Scholar, 7McGlynn P. Hunter C.N. Mol. Gen. Genet. 1993; 236: 227-234Crossref PubMed Scopus (25) Google Scholar, 8Lee J.K. Kaplan S. J. Bacteriol. 1992; 174: 1146-1157Crossref PubMed Google Scholar). The other class, represented by theR. capsulatus puc and crtI promoters and by theR. sphaeroides crtI promoter, has only a single CrtJ palindrome that is near or overlapping with the −10 or −35 regions (9Nickens D.G. Bauer C.E. J. Bacteriol. 1998; 180: 4270-4277Crossref PubMed Google Scholar, 10Elsen S. Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1998; 273: 30762-30769Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 11Lang H.P. Cogdell R.J. Gardiner A.T. Hunter C.N. J. Bacteriol. 1994; 176: 3859-3867Crossref PubMed Google Scholar). It is not clear why some promoters contain only one palindrome and others contain two. More detailed analysis of these promoters is needed to obtain a better understanding of aerobic repression of photosystem gene expression by CrtJ. In the present study, we utilized a combination of gel mobility shift and DNase I footprint analyses to demonstrate that CrtJ binding to the two palindromic sites in the bchC promoter region occurs in a cooperative fashion. DNA binding isotherms indicate that there is a 26-fold cooperativity of CrtJ binding to the −35 palindrome. The spacing of 8 base pairs (bp)1 between the twobchC palindromes is also shown to be critical for CrtJ binding. In a companion study (10Elsen S. Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1998; 273: 30762-30769Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), we demonstrate that aerobic repression of the puc and crtI promoters involves cooperative interactions among CrtJ bound to distant palindromes. Escherichia colistrain NM522 was used for routine cloning procedures (12Gaugh J.A. Murray N.E. J. Mol. Biol. 1983; 166: 1-19Crossref PubMed Scopus (344) Google Scholar). For overexpression of CrtJ, strain BL21(DE3) was utilized (13Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar). Plasmids pDAY23Ω (14Young D.A. Bauer C.E. Williams J.C. Marrs B.L. Mol. Gen. Genet. 1989; 218: 1-12Crossref PubMed Scopus (98) Google Scholar), pUC18 (15Vieira J. Messing J. Gene. 1982; 19: 259-268Crossref PubMed Scopus (3784) Google Scholar), and pET28::CrtJ (4Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1997; 272: 18391-18396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) have been described previously. Luria broth was used for agar-solidified plates and for liquid cultures (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Ampicillin and kanamycin were used at 100 μg/ml, and 30 μg/ml, respectively. His-tagged CrtJ was heterologously overexpressed in E. coli using a T7 RNA polymerase-based overexpression system as described previously (4Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1997; 272: 18391-18396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar,13Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar). Purification of His6-appended CrtJ from a 100-ml culture of cells was performed using nickel column affinity chromatography with all purification steps performed at 4 °C (Novagen). Fractions containing the highest CrtJ concentrations were pooled and dialyzed overnight in 1 liter of buffer composed of 50 mm Tris-HCl, pH 7.9, 50 mm potassium acetate, 1 mm EDTA, and 20% glycerol. The purified protein was then partitioned into 30-μl aliquots, subjected to rapid freezing using a dry ice-ethanol mixture, and stored at −80 °C. Protein concentrations were determined using the Bradford assay (17Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216412) Google Scholar) (Bio-Rad). The percent active fraction of CrtJ was determined with a nitrocellulose filter binding assay according to the method of Witherell and Uhlenbeck (18Witherell G.W. Uhlenbeck O.C. Biochemistry. 1989; 28: 71-76Crossref PubMed Scopus (91) Google Scholar) and Ponnampalam and Bauer (4Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1997; 272: 18391-18396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Several plasmids were constructed to separately clone the upstream (−30 to −47) and downstream palindromes (−4 to −21) from the bchC promoter region, as well as to introduce +6- and +11-bp insertions between the upstream and downstream palindromes. For these constructions, several segments of thebchC promoter region were generated by polymerase chain reaction (PCR) amplification and then cloned into appropriate cloning vectors. The individual PCRs contained 10 mm Tricine buffer (19Ponce M.R. Micol J.L. Nucleic Acids Res. 1992; 20: 623Crossref PubMed Scopus (95) Google Scholar), 0.3 mm dATP, 0.3 mm dGTP, 0.3 mm dTTP, 0.3 mm dCTP, 100 pmol of each primer, and 12.5 ng of BamHI-EcoRI-digested pDay23Ω template DNA (14Young D.A. Bauer C.E. Williams J.C. Marrs B.L. Mol. Gen. Genet. 1989; 218: 1-12Crossref PubMed Scopus (98) Google Scholar). The PCR cycle (PTC-100 Programmable Thermal Controller, MJ Research, Inc.) was initiated with a “hot start,” which involved preincubation of the PCR at 98 °C for 2.5 min followed by a reduction of reaction temperature to 90 °C, at which point, 1 unit of Taq RNA polymerase was added to the reaction buffer. Amplification was performed using 40 cycles of 20 s at 97 °C, 30 s at 62 °C, and 2 min at 72 °C. The reactions were then finished with a 20-min incubation period at 65 °C, and the resulting fragments were cloned into the TA cloning vector pCRII (Invitrogen). To PCR amplify a 87-bp fragment containing the upstream palindrome, primers BchC3 (5′-GTTCGGACCCGGCTTTGACC-3′) and BchC5 (5′ GGGGATCCGACTGTCAATTTGATTAGAC-3′) were used. For PCR amplification of 110-bp fragment containing the downstream palindrome, primers BchC2 (5′-CGGACATTATGACGACTTGCG-3′) and BchC4 (5′-CCGGATCCGGGCGTGTAAGTTCAATGATA-3′) were used. A 114-bp fragment, which also contains the downstream palindrome, was amplified using BchC2 in conjunction with primer BchC6 (5′-CGGATCCCAGTCGGGCGTGTAAGTTCAATGATA-3′). Each of the PCR products was cloned into the TA tailed site of pCRII (Invitrogen). Note that to facilitate subsequent subcloning steps,BamHI sites were engineered at the 5′-ends of oligonucleotides BchC4, BchC5, and BchC6 (underlined bases). Construction of +6- and +11-bp insertions between the palindromes involved first subcloning the amplified upstream palindrome from the vector pCRII as a 90-bp BamHI-EcoRI restriction fragment (the BamHI site came from the primer, and theEcoRI site came from the vector) into the vector pUC18 to construct the plasmid pUC3:5. For introducing a +6-bp insertion between the palindromes, we next subcloned the BchC2/BchC4 PCR-generated downstream palindrome fragment into pUC3:5. This was accomplished by subcloning a 146-bp BamHI-XhoI fragment from pCRII (the BamHI site was present in the primer, whereas theXhoI site was present in the vector) into theBamHI-SalI site of pUC3:5 vector, resulting in the vector pUC3:5–2:4. For constructing a +11-bp insertion between the palindromes, we subcloned the 151-bp BchC2/BchC6 PCR-generated downstream palindrome fragment from pCRII into pUC3:5 in a manner analogous to that described for the +6 insertion, generating the recombinant pUC3:5–2:6. Each of the final constructs were then sequenced to confirm the presence of +6 and +11 insertions between the palindromes. The upstream palindrome coded by the pCRII vector was amplified (as described above) using oligonucleotides BchC3 and 32P 5′-end-labeled primer pCRII-2 (5′-AGTCACGACGTTGTAAAACGA-3′), which hybridized to a region of the pCRII cloning vector that is located just outside of the multiple cloning site. Similarly, the downstream palindrome was amplified using two oligonucleotides, BchC2 and32P 5′-end-labeled pCRII-1 primer (5′-TTGTGAGCGGATAACAATTTCAC-3′), which also hybridized to the pCRII cloning vector. Oligonucleotide primers BchC2 (which was 5′-end-labeled) and BchC3 were used for amplification of the nativebchC promoter sequence from pDAY23Ω, as well as for amplification of the +6- and +11-bp additions that were coded by plasmids pUC3:5–2:4 and pUC3:5–2:6, respectively. 32P labeling of primers were carried at 37 °C for 1 h in a 10-μl reaction volume containing 100 pmol of primer, 1× T4 kinase buffer (New England Biolabs), 10 units of T4 polynucleotide kinase (New England Biolabs), and 320 μCi of [γ-32P]ATP (specific activity, 7000 Ci/mmol; Amersham Pharmacia Biotech). Unincorporated label was removed with a Sephadex G-50 fine Nick spin column (Amersham Pharmacia Biotech) per the manufacturer's instructions, followed by ethanol precipitation. The PCR-amplified fragment was then gel-purified, extracted from low melt agarose by hot (65 °C) phenol extraction, and concentrated by DEAE-Sephadex G-50 column chromatography. For gel mobility shift analysis, purified CrtJ was diluted into a 30-μl reaction buffer containing 9 fmol of32P-end-labeled DNA probe and 3 μl of a 10 mg/ml heparin solution (500-fold excess over the probe) in a reaction buffer composed of 50 mm Tris-HCl, pH 8.0, 1 mm EDTA, pH 8.1, 50 mm potassium acetate, 20% glycerol (v/v) (3Ponnampalam S.N. Buggy J.J. Bauer C.E. J. Bacteriol. 1995; 177: 2990-2997Crossref PubMed Google Scholar). Each reaction was then incubated for 20 min at 30 °C, loaded on a native 6% Tris-glycine-EDTA-buffered polyacrylamide gel, and electrophoresed at 16 mA for 2.5 h at 24 °C. The polyacrylamide gel was then dried and autoradiographed overnight at −80 °C with an intensifying screen. For footprint analysis, binding of purified CrtJ to the DNA fragment prior to DNase I digestion involved the similar conditions as described for the gel shift assays with the reaction mix composition containing 4 μl of end-labeled DNA (12 fmol), 82 μl of gel mobility shift buffer, and 9 μl of CrtJ at varying concentrations. After incubating the binding reaction at 30 °C for 20 min, 5 μl of DNase I (0.50 μg/ml) was added to the mixture, and the digestion was allowed to proceed for 2 min at room temperature. The reaction was then stopped by the addition of 100 μl of DNase I stop solution consisting of 0.6m ammonium acetate, 0.1 m EDTA, and 20 μg/ml calf thymus DNA followed by the addition of 200 μl of phenol:chloroform. The samples were vortexed, chilled on ice, and centrifuged for 5 min at 25 °C. DNA in the aqueous phase was then ethanol-precipitated by the addition of 500 μl of 100% ethanol, chilled for 10 min in a dry ice-ethanol bath, centrifuged for 10 min at 13,000 rpm, and then rinsed twice with 70% ethanol. The samples were then vacuum dried and resuspended in 6 μl of sequence dye mix composed of 80% deionized formamide, 1× TBE buffer (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar), 0.1% bromphenol blue, and 0.1% xylene cyanol. The samples were heated to 90 °C for 3 min and then resolved by electrophoresis on a 5% urea denaturing Long Ranger polyacrylamide gel in TBE buffer at a constant power of 60 W for 1 h. A modified Maxam and Gilbert G+A chemical sequencing reaction was used for determining the location of DNase I protection (20Maxam A.M. Gilbert W. Grossman L. Moldave K. Methods in Enzymology. 65. Academic Press, New York1980: 499-560Google Scholar). CrtJ DNA binding isotherms to the bchC promoter region were generated by DNase I footprint titration assays using 2-fold protein dilutions. The gel was then analyzed for CrtJ binding isotherms using a PhosphorImager (Molecular Dynamics) to quantitate the level of CrtJ protection of a single band in the upstream and downstream palindromes. Values were corrected for loading by normalization with a band from an unprotected region. We assayed for cooperative interactions between CrtJ bound to the −35 and −10 palindromes in the bchCpromoter by performing gel mobility shift and DNase I footprint analysis with a variety of DNA probes. Specific probes that were used included (i) a probe that contained the wild type promoter region flanked by 58 and 79 bp of upstream and downstream DNA, respectively (Fig. 2 A); (ii) a segment that contains only the −35 palindrome flanked by 58 and 134 bp of DNA (Fig. 2 B); and (iii) a segment that contains only the −10 palindrome flanked by 168 and 79 bp of DNA (Fig. 2 C). Similar to what we reported in a previous study (4Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1997; 272: 18391-18396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), we observed that the entire wild type bchC promoter probe exhibited a mobility shift when incubated with 0.25 and 0.50 μg of isolated CrtJ (Fig. 3, lanes 2 and 3). In contrast, there was no observable mobility shift using probes that only contain the −35 palindrome (Fig. 3, lanes 5–6) or only the −10 palindrome (Fig. 3, lanes 8–9) when using similar amounts of CrtJ. The absence of a discrete gel shift with probes containing only the −35 or −10 palindromes gives a qualitative indication that there is cooperative binding of CrtJ at the two palindromic sites in the wild type probe. DNase I footprint titration analyses were subsequently performed on the same probes used for gel mobility shift assays to quantitatively address cooperative interactions between CrtJ bound to the neighboring palindromes. As shown in Fig. 4(left panel), footprint analysis with the wild type probe exhibited protection of both palindromes at a CrtJ levels as low as 0.125 μg. In contrast, the probe that contains only the −35 palindrome (Fig. 4, middle panel) required approximately 2.0 μg of CrtJ to visualize protection of the palindromic region. No protection was observed on the −10 palindrome (Fig. 4, right panel) even at the highest level of CrtJ that was used (16 μg). In addition to providing additional evidence of cooperativity among CrtJ bound to the two palindromes, the footprint results also indicate that CrtJ has a higher affinity for the −35 palindrome than for the −10 palindrome. Isotherms of CrtJ binding to the −35 palindrome were generated by quantitating fractional protection of the −35 palindrome to DNase I digestion using a PhosphorImager as described previously (4Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1997; 272: 18391-18396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 21Brenowitz M. Senear D.F. Shea M.A. Ackers G.K. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8462-8466Crossref PubMed Scopus (146) Google Scholar). Values of fractional protection plotted against the logarithm of protein concentration (Fig. 5) produced curves with 26-fold different midpoint values of CrtJ protection of the −35 palindrome when in the presence or absence of the −10 palindrome. When corrected for percentage of active protein in the isolated CrtJ preparation (see under “Experimental Procedures” and Ref. 4Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1997; 272: 18391-18396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), we calculate an EC50 value of 7.8 × 10−8m for the −35 palindrome alone versus 0.3 × 10−8m for the −35 palindrome in the presence of the −10 palindrome. We next addressed whether the 8-bp spacing between the −35 and −10 palindromes is critical for cooperative interactions. For this analysis, we constructed probes in which the intervening sequences between the two bchC palindromes were increased from 8 to 14 bp (half-helical insertion) or from 8 to 19 bp (full-helical insertion) (Fig. 2, D and E, respectively). The results of gel mobility shift analysis with these probes indicate that increasing the spacing between the palindromes to 14 or 19 bp abolishes the formation of a stable DNA-CrtJ complex (Fig. 6, lanes 6–8 and 10–12, respectively). DNase I footprint titration analyses on these probes gave results similar to those observed with the half sites, namely, partial protection of the −35 palindrome at high input levels of CrtJ (>2 μg) and no protection of the −10 palindrome (data not shown). This indicates that the 8-bp spacing between the two palindromic sites is indeed important for proper cooperative binding of CrtJ to the bchC promoters. In conclusion, we have shown by both gel mobility shift and footprint analyses that CrtJ binds in a cooperative fashion to the two palindromic sites of the bchC promoter region. CrtJ binding to the −35 palindrome shows 26-fold cooperativity when spaced 8 nucleotides apart from the −10 palindrome. This is of the same order of magnitude of cooperative DNA binding that has been observed for other proteins. For example, in the PR promoter of bacteriophage lambda, cooperativity increases affinity of the λ repressor to the middle operator site (OR2) by 25-fold and to the right (OR1) operator site by 2-fold (22Ackers G.A. Shea M.A. Johnson A.D. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1129-1133Crossref PubMed Scopus (460) Google Scholar, 23Johnson A.D. Meyer B.J. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5061-5065Crossref PubMed Scopus (273) Google Scholar, 24Ptashne M. A Genetic Switch. Blackwell Scientific Publications, Boston, MA1987Google Scholar). CrtJ binding to the class of photosynthesis promoters that contain two closely spaced (7–8 bp) palindromes thus follows that of other well studied DNA-binding proteins that utilize cooperativity to facilitate binding. In the companion report by Elsen et al. (10Elsen S. Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1998; 273: 30762-30769Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), we have investigated the nature of CrtJ repression of photosynthesis promoters that contain only a single palindrome in the −10 to −35 promoter region. The Elsen et al. (10Elsen S. Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1998; 273: 30762-30769Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) study demonstrates that repression of this class of promoters requires the presence of a second distant (76–240 bp) palindrome. Thus, effective repression of target promoters by CrtJ requires cooperative interactions between CrtJ bound to two palindromes in each of the promoter regions that have been studied. We thank Dr. John Richardson for helpful advice and Dr. Terry Bird and Chen Dong for comments on the manuscript." @default.
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• W2068372232 title "Aerobic Repression of the Rhodobacter capsulatus bchCPromoter Involves Cooperative Interactions between CrtJ Bound to Neighboring Palindromes" @default.
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