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DNA Binding Characteristics of CrtJ

1997; Elsevier BV; Volume: 272; Issue: 29 Linguagem: Inglês

10.1074/jbc.272.29.18391

1083-351X

Stephen N. Ponnampalam, Carl E. Bauer,

Marine and coastal ecosystems

Previous genetic analysis indicated that the photosynthesis gene cluster from Rhodobacter capsulatuscoded for the transcription factor, CrtJ, that is responsible for aerobic repression of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression. In this study, we have heterologously overexpressed and purified CrtJ to homogeneity and shown by gel mobility shift assays that CrtJ is biologically active. DNase I footprint analysis confirms molecular genetic studies by showing that CrtJ binds to conserved palindromic sequences that overlap the −10 and −35 promoter regions of the bchC operon. Graphs of the percentage of DNA bound versus protein concentration show sigmoidal curves, which is highly indicative of cooperative binding of CrtJ to the two palindromic sites. A binding constant for interaction of CrtJ with the palindrome that spans the −10 region was calculated to be 4.8 × 10−9m, whereas affinity for the palindrome that spans the −35 region was found to be 2.9 × 10−9m. Binding of CrtJ to the bchCpromoter region was also found to be redox-sensitive, with CrtJ exhibiting a 4.5-fold higher binding affinity under oxidizingversus reducing conditions. Previous genetic analysis indicated that the photosynthesis gene cluster from Rhodobacter capsulatuscoded for the transcription factor, CrtJ, that is responsible for aerobic repression of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression. In this study, we have heterologously overexpressed and purified CrtJ to homogeneity and shown by gel mobility shift assays that CrtJ is biologically active. DNase I footprint analysis confirms molecular genetic studies by showing that CrtJ binds to conserved palindromic sequences that overlap the −10 and −35 promoter regions of the bchC operon. Graphs of the percentage of DNA bound versus protein concentration show sigmoidal curves, which is highly indicative of cooperative binding of CrtJ to the two palindromic sites. A binding constant for interaction of CrtJ with the palindrome that spans the −10 region was calculated to be 4.8 × 10−9m, whereas affinity for the palindrome that spans the −35 region was found to be 2.9 × 10−9m. Binding of CrtJ to the bchCpromoter region was also found to be redox-sensitive, with CrtJ exhibiting a 4.5-fold higher binding affinity under oxidizingversus reducing conditions. Rhodobacter capsulatus is a purple nonsulfur photosynthetic bacterium that regulates synthesis of its photosynthetic apparatus in response to alterations in oxygen tension and light intensity. Oxygen tension at atmospheric levels (21%) results in virtual suppression of photopigment production, whereas reduced oxygen tension ( 95% as judged by Coomassie Blue staining;lane 4) by affinity binding of the His6-tagged CrtJ to a Ni2+ column. Gel retardation (mobility shift) assays were subsequently performed to ascertain if isolated CrtJ was active. As a control, we performed a gel retardation assay with crude cell lysates obtained from wild type and CrtJ-disrupted R. capsulatus cells. In confirmation of previous results (11Ponnampalam S.N. Buggy J.J. Bauer C.E. J. Bacteriol. 1995; 177: 2990-2997Crossref PubMed Google Scholar), we observed that a DNA fragment that contains the bchC promoter region was shifted to a reduced electrophoretic mobility when incubated with the wild type extract (Fig. 2, lane 2). This mobility shift is absent in extracts derived from the crtJ-disrupted strain DB469 (lane 3), suggesting that the mobility shift observed with the wild type extract resulted from an interaction of CrtJ with the bchC promoter region. This supposition is supported by mobility shifts obtained with increasing concentrations of purified CrtJ (lanes 4–6) that has an electrophoretic mobility identical to that observed with wild type crude extracts. These results indicate that heterologously expressed CrtJ was isolated in a properly folded form and that it has specificity for a sequence within thebchC promoter region. DNase I protection (footprint) analysis of CrtJ binding to the bchC promoter region was performed on both DNA strands by selectively 5′-end labeling either the top or bottom strand. As seen from the DNase I digestion patterns in Fig. 3, CrtJ protects a region of the top strand ranging from −3 to −52 and on the bottom strand from −1 to −49. Inspection of the protected DNA sequence (Fig. 3) indicates that this region contains two copies of the palindromic sequence TGT-N4-TCAA-N3-ACA, one of which flanks the −10 and the other the −35 region of thebchC promoter (11Ponnampalam S.N. Buggy J.J. Bauer C.E. J. Bacteriol. 1995; 177: 2990-2997Crossref PubMed Google Scholar, 12Ma D. Cook D.N. O'Brien D.A. Hearst J.E. J. Bacteriol. 1993; 175: 2037-2045Crossref PubMed Google Scholar, 24Brenowitz 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). Several sites hypersensitive to DNase I digestion are also observed on the top and bottom strands (Figs. 3 and 4). These hypersensitive strands extend 36 base pairs upstream and 58 base pairs downstream from the region of CrtJ protection.Figure 4Summary of the top and bottom strand protection patterns of CrtJ binding to the bchC promoter region. The protected regions of both strands are indicated bybrackets. Hypersensitive sites are represented byshort vertical arrows; vertical arrows above theDNA sequence represent hypersensitive sites seen on the top strand, and arrows below the sequence represent hypersensitive sites seen on the bottom strand. The −10 and −35 promoter regions are underlined. The white on black sequences with half-arrows denote the palindromic sites. The large thick arrow denotes the start and direction of transcription of the bchC operon.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The fraction of CrtJ that is active in DNA binding was determined according to the filter binding method of Witherell and Uhlenbeck (25Witherell G.W. Uhlenbeck O.C. Biochemistry. 1989; 28: 71-76Crossref PubMed Scopus (91) Google Scholar), which involves quantitating the amount of maximal DNA probe bound with a known amount of protein. If the oligomerization state of the protein bound to the DNA probe is known, then it is possible to calculate the percentage of fraction of protein that is active. For our analysis, the CrtJ protein concentration was held constant at 1.2 pm (at approximately half saturation) with the 32P-labeled bchC DNA probe varied in the vicinity of the protein (CrtJ) concentration. As shown in the DNA titration curve in Fig. 5, the amount of DNA bound by the filter rises to a saturation point of 0.11 pmol of DNA (note that this value has been corrected for the efficiency of probe retention by the filter as described under “Materials and Methods”). Assuming that the DNA probe maximally binds to the filter when both palindromes are fully occupied and that two CrtJ bind per palindrome for a total of four CrtJ/probe, then at saturating levels of input DNA bound (0.11 pmol) there must be 0.44 pmol of protein bound by the filter. Thus, the fraction of protein that is active corresponds to 0.44 pmol of protein retained by the filter/1.2 pmol of input protein in the assay, which corresponds to an active protein fraction of 36.7%. Binding isotherms for interactions of CrtJ to each of the individual palindromes were determined using a DNase I footprint titration assay according to the method of Brenowitz et al.(24Brenowitz 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). This method involves quantitating, by PhosphorImager analysis, CrtJ-mediated protection of a DNase I digestion site in each of the palindromes using small increments of CrtJ and correcting for background. The optical density ratios are then converted to fractional protection values. However, because even at saturating levels of protein, the DNA is not completely protected from DNase I digestion, the fractional protection values have to be converted to fractional saturation values. The fractional saturation values are then plotted against the logarithm of the protein concentration (Fig.6). As shown in Fig. 6, CrtJ protection of the upstream (−21 to −52) and downstream (−1 to −20) palindromes exhibits sigmoidal curves of protection with an average Hill coefficient of 2.5 for each binding site. Since the Hill coefficient is greater than 2, it indicates that there are cooperative interactions among three or more protein subunits that are binding to the two palindromic binding sites. This further supports the hypothesis that CrtJ binds in a multimeric form, possibly as a dimer to each palindromic site and that tetramerization of these dimers induces a cooperative interaction (see “Discussion”). From the fractional saturation values, we can also determine an EC50 value for binding of CrtJ to the upstream and downstream palindromes. The values obtained, after correcting for 37% active protein, are values of 2.9 × 10−9m for binding to the upstream palindrome and 4.8 × 10−9m for binding to the downstream palindrome. Since previous genetic analysis indicated that CrtJ most likely functions as an aerobic repressor (11Ponnampalam S.N. Buggy J.J. Bauer C.E. J. Bacteriol. 1995; 177: 2990-2997Crossref PubMed Google Scholar), we next addressed whether binding of CrtJ to the bchC promoter was affected by alterations in the redox state of the binding buffer. For this analysis, we first preincubated CrtJ for 30 min in the presence of a binding buffer that contained soluble redox mediators (to facilitate the transport of electrons from the oxidizing/reducing agent to the protein center) (22Bauer C.E. Hesse S.D. Gumport R.I. Gardner J.F. J. Mol. Biol. 1986; 192: 513-527Crossref PubMed Scopus (35) Google Scholar) as well as various oxidizing/reducing agents. After preincubation, a 32P-end-labeled bchC DNA probe was added to the samples, incubated for an additional 20 min, and then subjected to gel electrophoresis. As shown in the gel shift assays in Fig. 7, preincubation of CrtJ in the presence of a binding buffer that was saturated with oxygen (by bubbling molecular oxygen into the binding buffer) or 20 mm potassium ferricyanide, which have redox values of approximately +800 and +450 mV, respectively, promoted excellent binding conditions. In contrast, preincubation of CrtJ under conditions where oxygen was replaced with the redox neutral gas argon or with 10 mm of the strong reducing reagent sodium dithionite (approximately −600 mV) resulted in a significant reduction of binding activity. Three separate reactions were performed to determine if the observed redox response was reversible. In the first reaction, CrtJ was incubated separately with either 10 mm sodium dithionite or 20 mm potassium ferricyanide for 1 h. In a second reaction, CrtJ was first incubated for half an hour with 10 mm sodium dithionite, an excess of potassium ferricyanide (20 mm) was added, and then the reaction was incubated for a further 30 min. As shown in Fig. 8, the inhibitory effect of reducing conditions is indeed reversible, since the addition of the excess potassium ferricyanide to the CrtJ sample that had been inhibited with dithionite promoted the formation of a stable DNA-protein complex in the gel mobility shift assay (lanes 8and 9). Thus, CrtJ appears to be capable of sensing environmental redox values and adjusting binding accordingly. We next performed gel mobility shift assays with small increments (0.05–0.10 μg) of CrtJ to generate binding isotherms for the determination of EC50 values (effective concentration of CrtJ for 50% response) of CrtJ binding to the bchC promoter region under varying redox conditions. Cooperative binding of CrtJ to the two palindromic sites of the bchC promoter region is also revealed when the percentage of DNA bound is plotted against protein concentration. Sigmoidal curves were obtained indicating cooperativity between the two binding sites (Fig. 9). The EC50 value for CrtJ binding to the bchCpromoter region indicates that CrtJ binding affinity for thebchC promoter increases 4.5-fold under oxidizingversus reducing conditions (Table I). The corrected EC50 values for binding of CrtJ to thebchC promoter region (Table I), as based on gel mobility shifts (the values of which were corrected for percentage of active fraction and for the binding of four CrtJ/promoter fragment), indicates that binding to the promoter fragment is 6.0 × 10−9m under oxidizing conditions (oxygen) and 2.7 × 10−8m under reducing conditions (sodium dithionite). Intermediate EC50 values were obtained for less severe oxidizing/reducing conditions. These values are 8.4 × 10−9m and 1.9 × 10−8m for potassium ferricyanide and argon, respectively (TableI).Table IBinding constants between DNA and purified CrtJ under various redox conditions as calculated by gel mobility shift analysisRedox reagentEC50Corrected EC50 valueμmmOxygen0.0656.0 × 10−9Potassium ferricyanide0.0928.4 × 10−9Argon0.211.9 × 10−8Sodium dithionite0.292.7 × 10−8 Open table in a new tab Our results demonstrate that highly purified His-tagged CrtJ can be readily isolated in an active form by a one-step purification using a Ni2+ charged column. The isolated protein is active as evidenced from gel mobility shift assays where purified CrtJ shows an identical shift with that of the crude lysate of wild type R. capsulatus cells and by the results of our DNase I footprint titration assays. The protection pattern of CrtJ binding to both the top and bottom strands of the bchC promoter region shows an area of protection extending approximately 52 base pairs from −1 to −51. This region contains two conserved palindromic sequences TGT-N12-ACA that are centered around the ς70-like promoter sequences present at the −35 and −10 regions of the bchC operon (11Ponnampalam S.N. Buggy J.J. Bauer C.E. J. Bacteriol. 1995; 177: 2990-2997Crossref PubMed Google Scholar, 12Ma D. Cook D.N. O'Brien D.A. Hearst J.E. J. Bacteriol. 1993; 175: 2037-2045Crossref PubMed Google Scholar, 26Wellington C.L. Beatty J.T. Gene ( Amst. ). 1989; 83: 251-261Crossref PubMed Scopus (13) Google Scholar). Recognition of this palindrome by CrtJ is also supported by prior mutational analysis, which indicated that they were involved in the binding of an aerobic repressor (12Ma D. Cook D.N. O'Brien D.A. Hearst J.E. J. Bacteriol. 1993; 175: 2037-2045Crossref PubMed Google Scholar). Evidence at hand indicates that cooperative interactions occur between CrtJ bound at the two palindromes. Cooperativity is indicated by the formation of sigmoidal curves and by the Hill coefficient obtained for CrtJ binding to the two palindromic sites, which indicates that this interaction involves more than 2.5 CrtJ polypeptides. We also have unpublished data that indicates that CrtJ binds very poorly to DNA segments that contain only one of the two identified bchCpalindromes. 2S. Ponnampalam and C. Bauer, unpublished results. Given well characterized interactions observed among other prokaryotic transcription factors such as the λcI repressor, the nitrogen regulator NtrC, and the lacI repressor in E. coli (27Ptashne M. A Genetic Switch: Gene Control and Phage λ. Cell and Blackwell Scientific Publications, Cambridge, MA1986: 87-93Google Scholar, 28Weiss V. Claverie-Martin F. Magasanik B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5088-5092Crossref PubMed Scopus (115) Google Scholar, 29Lewis M. Chang 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 most likely scenario is that CrtJ binds as a dimer to each of the palindromes and that the dimers cooperatively interact to form stable tetramers. The EC50 values of CrtJ binding to the bchCpromoter region under oxidizing conditions is 6.0 × 10−9m, as based on gel mobility shift results, which is the same order of magnitude as that of the λcI repressor binding to the right operator site, OR1 (under “physiological conditions”, cI repressor has a K d of 3 × 10−9m for OR1 (13Sauer, R. T. (1979) Molecular Characterization of the Repressor and Its Gene cI. Ph.D. thesis, Harvard University.Google Scholar, 30Johnson A. Meyer B.J. Ptashne M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5061-5065Crossref PubMed Scopus (272) Google Scholar, 31Johnson A.D. Pabo C.O. Sauer R.T. Methods Enzymol. 1980; 65: 839-856Crossref PubMed Scopus (115) Google Scholar)). This value is, however, several orders of magnitude lower than that observed for other repressors, such as the lac repressor, which has a binding affinity of 10−12m (32Lin S. Riggs A.D. Cell. 1975; 4: 107-111Abstract Full Text PDF PubMed Scopus (229) Google Scholar). The modest affinity of CrtJ binding to the bchC promoter may reflect the fact that CrtJ-regulated promoters are only moderately repressed (1.5–2-fold) by CrtJ (11Ponnampalam S.N. Buggy J.J. Bauer C.E. J. Bacteriol. 1995; 177: 2990-2997Crossref PubMed Google Scholar, 12Ma D. Cook D.N. O'Brien D.A. Hearst J.E. J. Bacteriol. 1993; 175: 2037-2045Crossref PubMed Google Scholar), which is contrasted by the 1,000-fold (32Lin S. Riggs A.D. Cell. 1975; 4: 107-111Abstract Full Text PDF PubMed Scopus (229) Google Scholar) repression exhibited by the lac repressor. Since previous genetic analysis indicated that CrtJ functions as an aerobic repressor (8Penfold R.J. Pemberton J.M. Curr. Microbiol. 1991; 23: 259-263Crossref Scopus (30) Google Scholar, 9Penfold R.J. Pemberton J.M. J. Bacteriol. 1994; 176: 2869-2876Crossref PubMed Google Scholar, 10Gomelsky M. Kaplan S. J. Bacteriol. 1995; 177: 1634-1637Crossref PubMed Google Scholar, 11Ponnampalam S.N. Buggy J.J. Bauer C.E. J. Bacteriol. 1995; 177: 2990-2997Crossref PubMed Google Scholar, 12Ma D. Cook D.N. O'Brien D.A. Hearst J.E. J. Bacteriol. 1993; 175: 2037-2045Crossref PubMed Google Scholar), it was perhaps not surprising to find that CrtJ binds to the bchC DNA fragment better under oxidizingversus reducing conditions. The calculated EC50value of CrtJ binding to the bchC promoter region under oxidizing conditions is 4.5-fold higher than that observed under reducing conditions, suggesting that CrtJ may have redox sensing capabilities. The effect of altering the redox state on CrtJ binding is reversible, suggesting that reducing conditions are not nonspecifically affecting DNA binding activity. Furthermore, CrtJ binding is best under highly oxidizing conditions (such as oxygen-saturated binding buffer), which is a condition that inhibits binding of other redox-responding DNA binding proteins such as FNR, DtxR, SoxR, and Fur. These latter redox-responding proteins have oxygen-labile iron or iron-sulfur clusters that are required for optimal DNA binding capabilities (32Lin S. Riggs A.D. Cell. 1975; 4: 107-111Abstract Full Text PDF PubMed Scopus (229) Google Scholar, 33Bagg A. Neilands J.B. Biochemistry. 1987; 26: 5471-5477Crossref PubMed Scopus (424) Google Scholar, 34Hidalgo E. Demple B. EMBO J. 1994; 1: 138-146Crossref Scopus (247) Google Scholar, 35Khoroshilova N. Beinert H. Kiley P.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2499-2503Crossref PubMed Scopus (173) Google Scholar, 36Schmitt M.P. Twiddy E.M Holmes R.H. Proc. Natl. Sci. U. S. A. 1992; 89: 7576-7580Crossref PubMed Scopus (64) Google Scholar, 37Schmitt M.P. Holmes R.K. Mol. 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Bacteriol. 1997; 179: 128-134Crossref PubMed Google Scholar) indicates that in Rhodobacter sphaeroides a second protein (AppA) may also be involved in controlling redox-sensitive binding by CrtJ. Although no experimental evidence has been obtained for a function for AppA in controlling CrtJ activity, it is possible that AppA may be sensing redox and somehow transmitting this information to CrtJ. A homolog for AppA in R. capsulatus has not yet been described, so at this time we are unable to biochemically ascertain whether AppA affects the in vitro redox response of CrtJ DNA binding. Finally, there are several studies that indicate that CrtJ may be a global repressor of pigment biosynthesis genes and that its presence is conserved in diverse species of anoxygenic photosynthetic bacteria. For example, sequence and genetic analysis of the R. capsulatusphotosynthesis gene cluster indicates the presence of similar palindromic motifs within putative promoter sequences for bacteriochlorophyll, carotenoid, and light harvesting-II structural genes (2Bauer C.E. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria: Regulation of Photosynthesis Gene Expression. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 1221-1234Google Scholar, 12Ma D. Cook D.N. O'Brien D.A. Hearst J.E. J. Bacteriol. 1993; 175: 2037-2045Crossref PubMed Google Scholar, 20Alberti M. Burke D.E. Hearst J.E. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 1083-1106Google Scholar, 26Wellington C.L. Beatty J.T. Gene ( Amst. ). 1989; 83: 251-261Crossref PubMed Scopus (13) Google Scholar, 42Armstrong G.A. Alberti M. Leach F. Hearst J.E. Mol. & Gen. Genet. 1989; 216: 254-268Crossref PubMed Scopus (263) Google Scholar, 43McGlynn P. Hunter C.N. Mol. & Gen. Genet. 1993; 236: 227-234Crossref PubMed Scopus (23) Google Scholar). Sequence analysis of the pucpromoter from such diverse species as R. sphaeroides (44Lee J.K. Kiley P.J. Kaplan S. J. Bacteriol. 1989; 171: 3391-3405Crossref PubMed Google Scholar),R. capsulatus (45Zucconi A.P. Beatty J.T. J. Bacteriol. 1988; 170: 877-882Crossref PubMed Google Scholar), and Rhodopseudomonas palustris (46Tadros M.H. Katsiou E. Hoon M.A. Yurkova N. Ramji D.P. Eur. J. Biochem. 1993; 217: 867-875Crossref PubMed Scopus (45) Google Scholar) also shows the presence of the palindrome that is characterized in this study, indicating that CrtJ may be conserved among a diverse group of anoxygenic photosynthetic bacteria. Thus, any insights into the mechanism of redox responsiveness of CrtJ binding to photosystem promoters could also provide insight into how these additional organisms control photosystem development. We thank Dr. John Richardson for helpful comments regarding this study.