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• W2036822952 abstract "CooA, a member of the cAMP receptor protein (CRP) family, is a CO-sensing transcription activator fromRhodospirillum rubrum that binds specific DNA sequences in response to CO. The location of the CooA-binding sites relative to the start sites of transcription suggested that the CooA-dependent promoters are analogous to class II CRP-dependent promoters. In this study, we developed anin vivo CooA reporter system in Escherichia coli and an in vitro transcription assay using RNA polymerases (RNAP) from E. coli and from Rhodobacter sphaeroides to study the transcription properties of CooA and the protein-protein interaction between CooA and RNAP. The ability of CooA to activate CO-dependent transcription in vivoin heterologous backgrounds suggested that CooA is sufficient to direct RNAP to initiate transcription and that no other factors are required. This hypothesis was confirmed in vitro with purified CooA and purified RNAP. Use of a mutant form of E. coli RNAP with α subunits lacking their C-terminal domain (α-CTD) dramatically decreased CooA-dependent transcription of the CooA-regulated R. rubrum promoter PcooF in vitro, which indicates that α-CTD plays an important role in this activation. DNase I footprinting analysis showed that CooA facilitates binding of wild-type RNAP, but not α-CTD-truncated RNAP, to PcooF. This facilitated binding provides evidence for a direct contact between CooA and α-CTD of RNAP during activation of transcription. Mapping the CooA-contact site in α-CTD suggests that CooA is similar but not identical to CRP in terms of its contact sites to the α-CTD at class II promoters. CooA, a member of the cAMP receptor protein (CRP) family, is a CO-sensing transcription activator fromRhodospirillum rubrum that binds specific DNA sequences in response to CO. The location of the CooA-binding sites relative to the start sites of transcription suggested that the CooA-dependent promoters are analogous to class II CRP-dependent promoters. In this study, we developed anin vivo CooA reporter system in Escherichia coli and an in vitro transcription assay using RNA polymerases (RNAP) from E. coli and from Rhodobacter sphaeroides to study the transcription properties of CooA and the protein-protein interaction between CooA and RNAP. The ability of CooA to activate CO-dependent transcription in vivoin heterologous backgrounds suggested that CooA is sufficient to direct RNAP to initiate transcription and that no other factors are required. This hypothesis was confirmed in vitro with purified CooA and purified RNAP. Use of a mutant form of E. coli RNAP with α subunits lacking their C-terminal domain (α-CTD) dramatically decreased CooA-dependent transcription of the CooA-regulated R. rubrum promoter PcooF in vitro, which indicates that α-CTD plays an important role in this activation. DNase I footprinting analysis showed that CooA facilitates binding of wild-type RNAP, but not α-CTD-truncated RNAP, to PcooF. This facilitated binding provides evidence for a direct contact between CooA and α-CTD of RNAP during activation of transcription. Mapping the CooA-contact site in α-CTD suggests that CooA is similar but not identical to CRP in terms of its contact sites to the α-CTD at class II promoters. CooA is a CO-sensing transcription activator fromRhodospirillum rubrum, and it activates the expression of the cooFSCTJ and cooMKLXUH operons in response to CO (1Shelver D. Kerby R.L. He Y. Roberts G.P. J. Bacteriol. 1995; 177: 2157-2163Crossref PubMed Google Scholar, 2He Y. Shelver D. Kerby R.L. Roberts G.P. J. Biol. Chem. 1996; 271: 120-123Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 3Fox J.D. He Y. Shelver D. Roberts G.P. Ludden P.W. J. Bacteriol. 1996; 178: 6200-6208Crossref PubMed Google Scholar). These two adjacent operons encode proteins that oxidize CO to CO2 with concomitant reduction of H+ to H2 and allow growth of this organism on CO as a sole energy source (4Bonam D. Lehman L. Roberts G.P. Ludden P.W. J. Bacteriol. 1989; 171: 3102-3107Crossref PubMed Google Scholar, 5Kerby R.L. Hong S.S. Ensign S.A. Coppoc L.J. Ludden P.W. Roberts G.P. J. Bacteriol. 1992; 174: 5284-5294Crossref PubMed Google Scholar, 6Kerby R.L. Ludden P.W. Roberts G.P. J. Bacteriol. 1995; 177: 2241-2244Crossref PubMed Google Scholar). cooA lies immediately 3′ of thecooFSCTJ operon, and its expression does not depend on CO. cooA insertion and deletion mutants fail to accumulate mRNA from either the cooFSCTJ or cooMKLXUHoperons in response to CO (1Shelver D. Kerby R.L. He Y. Roberts G.P. J. Bacteriol. 1995; 177: 2157-2163Crossref PubMed Google Scholar, 2He Y. Shelver D. Kerby R.L. Roberts G.P. J. Biol. Chem. 1996; 271: 120-123Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Sequence analysis suggested that CooA is a homolog of cAMP receptor protein (CRP), 1The abbreviations used are: CRP, cAMP receptor protein; RNAP, RNA polymerase; α-CTD, the C-terminal domain of the α subunit; AR1, activating region 1; AR2, activating region 2; AR3, activating region 3; UP elements, A + T-rich sequences located upstream in certain promoters that increase promoter strength; X-gal, 5-bromo-4-chloro-3-indolylβ-d-galactopyranoside. a well characterized transcription factor in Escherichia coli (1Shelver D. Kerby R.L. He Y. Roberts G.P. J. Bacteriol. 1995; 177: 2157-2163Crossref PubMed Google Scholar). The proposed helix-turn-helix DNA binding motif of CooA is highly similar to that of CRP, whereas the effector-binding domain displays lower similarity with CRP. CooA is not abundant in R. rubrum, but overexpression and purification of CooA were achieved in both R. rubrum andE. coli. CooA purified anaerobically from R. rubrum was indistinguishable spectroscopically or in terms of activity from CooA purified aerobically from either R. rubrum or E. coli (7Shelver D. Kerby R.L. He Y. Roberts G.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11216-11220Crossref PubMed Scopus (192) Google Scholar, 8Shelver D. Reynolds M.F. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Parks R.B. Burstyn J.N. Roberts G.P. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar). CooA is homodimeric under all conditions and contains approximately 2 mol of protoheme/dimer. CO directly binds to the heme of the reduced form of CooA, but not to the oxidized form, and this CO binding induces a conformational change in CooA that allows it to bind DNA in a sequence-specific manner (8Shelver D. Reynolds M.F. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Parks R.B. Burstyn J.N. Roberts G.P. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar). Oxidized CooA can be readily reduced, acquiring the ability to bind CO, and the mechanism for this reversible redox reaction involves an unusual switch of protein ligands on one side of the heme (8Shelver D. Reynolds M.F. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Parks R.B. Burstyn J.N. Roberts G.P. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar, 9Aono S. Ohkubo K. Matsuo T. Nakajima H. J. Biol. Chem. 1998; 273: 25757-25764Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Because the heme of CooA is 6-coordinate when reduced and when bound by CO, CO binding apparently displaces one of the axial ligands to the heme. It is our working hypothesis that this protein ligand displacement serves as the trigger for the conformational change in the CooA structure upon CO binding (8Shelver D. Reynolds M.F. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Parks R.B. Burstyn J.N. Roberts G.P. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar, 9Aono S. Ohkubo K. Matsuo T. Nakajima H. J. Biol. Chem. 1998; 273: 25757-25764Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). E. coli RNAP is composed of α2ββ′ς subunits; the role of ς is to bind −10 and −35 promoter elements. At promoters with A + T-rich sequences upstream of the −35 hexamer, termed “UP elements,” the C-terminal domain of the α subunit (α-CTD) makes specific DNA contacts that enhance transcription initiation (13Ross W. Gosink K.K. Salomon J. Igarashi K. Zou C. Ishihama A. Severinov K. Gourse R.L. Science. 1993; 262: 1407-1413Crossref PubMed Scopus (622) Google Scholar). Two regions of the α-CTD (including residues Leu262, Arg265, Asn268, Cys269, Gly296, Lys298, and Ser299) were found to be important for UP element-dependent transcription and DNA binding (14Gaal T. Ross W. Blatter E. Tang H. Jia X. Krishnan V.V. Assa-Munt N. Ebright R.H. Gourse R.L. Genes Dev. 1996; 10: 16-26Crossref PubMed Scopus (170) Google Scholar, 15Murakami K. Fujita N. Ishihama A. EMBO J. 1996; 15: 4358-4367Crossref PubMed Scopus (105) Google Scholar). The α-CTD is an independently folding domain of RNAP that is joined to the N-terminal domain of the α subunit by a flexible linker that allows the α-CTD to occupy different positions at different promoters (10Blatter E. Ross W. Tang H. Gourse R.L. Ebright R. Cell. 1994; 78: 889-896Abstract Full Text PDF PubMed Scopus (197) Google Scholar, 11Busby S. Ebright R.H. Cell. 1994; 79: 743-746Abstract Full Text PDF PubMed Scopus (350) Google Scholar, 12Jeon Y.H. Negishi T. Shirakawa M. Yamazaki T. Fujita N. Ishihama A. Kyogoku Y. Science. 1995; 270: 1495-1497Crossref PubMed Scopus (153) Google Scholar). The α-CTD is also known to make specific contacts with a range of activator proteins (16Ebright R.H. Busby S. Curr. Opin. Genet. Dev. 1995; 5: 197-203Crossref PubMed Scopus (136) Google Scholar, 17Ishihama A. Mol. Microbiol. 1992; 6: 3283-3288Crossref PubMed Scopus (138) Google Scholar). E. coli CRP controls the transcription of numerous genes involved in carbon source utilization. Upon the binding of its allosteric effector, cAMP, CRP undergoes a conformational change that allows a dimer of CRP to bind to a specific 22-base pair sequence at target promoters (consensus sequence is 5′-AAATGTGATCTAGATCACATTT-3′, in which the most important bases for CRP recognition are in bold) and to activate transcription at those promoters (18Kolb A. Busby S. Buc H. Garges S. Adhya S. Annu. Rev. Biochem. 1993; 62: 749-795Crossref PubMed Google Scholar). CRP-dependent promoters can be grouped into two classes based on the position of the CRP-binding site relative to the start of transcription as well as on the mechanism for transcription activation (19Ebright R. Mol. Microbiol. 1993; 8: 797-802Crossref PubMed Scopus (168) Google Scholar). At class I promoters, the DNA-binding site for CRP is upstream of that for RNAP and is centered at position −61.5, −71.5, −82.5, or −92.5. At class II CRP-dependent promoters, to which the CooA-dependent promoters are analogous, the binding site for CRP is centered at −41.5, overlapping the −35 region, and the α-CTD binds to DNA upstream of the CRP dimer. Direct interaction between CRP and RNAP plays a pivotal role in transcription activation at both promoter classes (20Busby S. Ebright R.H. Mol. Microbiol. 1997; 23: 853-859Crossref PubMed Scopus (159) Google Scholar, 21Dove S.L. Joung J.K. Hochschild A. Nature. 1997; 386: 627-630Crossref PubMed Scopus (228) Google Scholar). In particular, transcription activation at class II promoters requires two distinct contacts between CRP and the α subunit and a third contact between CRP and ς70. One interaction is between activating region 1 (AR1) of the upstream subunit of the CRP dimer and the α-CTD. This interaction increases initial binding of RNAP to the promoter (22Zhou Y. Pendergrast P.S. Bell A. Williams R. Busby S. Ebright R. EMBO J. 1994; 13: 4549-4557Crossref PubMed Scopus (81) Google Scholar). Recently, residues 285–288 and 317 of α-CTD have been shown to comprise the surface that interacts with AR1 of CRP at class II promoters (23Savery N.J. Lloyd G.S. Kainz M. Gaal T. Ross W. Ebright R.H. Gourse R.L. Busby S. EMBO J. 1998; 17: 3439-3447Crossref PubMed Scopus (118) Google Scholar). The second contact, between activating region 2 (AR2) of the downstream subunit of CRP and the N-terminal domain of the α subunit, facilitates isomerization of the closed complex to the open complex (24Niu W. Kim Y. Tau G. Heyduk T. Ebright R. Cell. 1996; 87: 1123-1134Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The residues in AR1 and AR2 of CRP are not conserved in CooA, which suggests that there might be certain differences in the interactions of CooA and CRP with α. The third activator region (AR3) in CRP, formed by residues 52–58, interacts with ς70 of RNAP (20Busby S. Ebright R.H. Mol. Microbiol. 1997; 23: 853-859Crossref PubMed Scopus (159) Google Scholar). Because the AR3 region in CRP is highly similar to an analogous region in CooA, this region in CooA might serve an AR3-like function. The two CooA-regulated R. rubrum promoters, PcooFand PcooM, contain 2-fold symmetric DNA sequences that serve as CooA-binding sites and are similar to the CRP consensus sequence. This is consistent with the similarity between CooA and CRP in their helix-turn-helix motifs (2He Y. Shelver D. Kerby R.L. Roberts G.P. J. Biol. Chem. 1996; 271: 120-123Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 3Fox J.D. He Y. Shelver D. Roberts G.P. Ludden P.W. J. Bacteriol. 1996; 178: 6200-6208Crossref PubMed Google Scholar). The CooA-binding sites lie at the −43.5 and −38.5 positions relative to the transcription start sites in PcooF and PcooM, respectively, overlapping with the −35 region (2He Y. Shelver D. Kerby R.L. Roberts G.P. J. Biol. Chem. 1996; 271: 120-123Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 3Fox J.D. He Y. Shelver D. Roberts G.P. Ludden P.W. J. Bacteriol. 1996; 178: 6200-6208Crossref PubMed Google Scholar). This overlap suggests that both CooA-regulated promoters are analogous to class II CRP-dependent promoters. We chose PcooF for this study because it is the stronger promoter based on the amount of primer extension product and level of coo-encoded proteins synthesized in vivo(1Shelver D. Kerby R.L. He Y. Roberts G.P. J. Bacteriol. 1995; 177: 2157-2163Crossref PubMed Google Scholar, 2He Y. Shelver D. Kerby R.L. Roberts G.P. J. Biol. Chem. 1996; 271: 120-123Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Although CooA shares some common features with CRP, such as DNA binding properties and effector-induced activation, it displays striking differences from CRP in the effector-binding domain and in regions AR1 and AR2. We were interested to know whether CooA was necessary and sufficient for CO-dependent activation of transcription and whether the mechanism of activation by CooA was similar to that of CRP. In this work, we used in vivo CooA reporter systems andin vitro transcription assays to examine the properties of CooA in transcription activation. Because of the particular questions we wished to address, we performed the bulk of the work with RNAP fromE. coli, so that any differences between CooA and the CRP detected would reflect properties of CooA. The nature of transcription activation by CooA was investigated through the study of the interaction between CooA and RNAP and, in particular, the interaction between CooA and the α-CTD. CooA was overexpressed from a vector, pYHA1, created as follows. A 1.9-kilobasePvuI-BamHI fragment containing Ptac-cooA-rrnBT1T2, was isolated from pKK223-3 (8Shelver D. Reynolds M.F. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Parks R.B. Burstyn J.N. Roberts G.P. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar), digested with PvuI, mung bean nuclease, and BamHI, and cloned into theEcoRV and BamHI sites of pACYC184. A plasmid containing a PcooF-lacZ fusion, pYHF4, was constructed by inserting a polymerase chain reaction-amplifiedEcoRI-HindIII fragment, extending from positions −250 to +70 of PcooF, into plasmid pMSB1 (25Rao L. Ross W. Appleman J.A. Gaal T. Leirmo S. Schlax P.J. Record M.T. Gourse R.L. J. Mol. Biol. 1994; 235: 1421-1435Crossref PubMed Scopus (185) Google Scholar), which created pMSBPcooF. The reporter region was recombined from plasmid pMSBPcooF into λ phage RS468 (26Simons R.W. Houman F. Kleckner N. Gene. 1987; 53: 85-96Crossref PubMed Scopus (1301) Google Scholar) in strain DH5α containing pYHA1. Lysogens were screened by the blue color of plaques on Luria broth (LB) + X-gal plates incubated anaerobically in the presence of CO. The promoter region of integrated PcooF-lacZfusion in the chromosome was confirmed by DNA sequencing. Polymerase chain reaction-amplified EcoRI-HindIII fragments from positions −250 to +70, −90 to +70, and −60 to +70 of PcooFwere cloned into pRLG770, which contains transcription terminatorrrnBT1T2 downstream of the multicloning site (27Ross W. Thompson J.F. Newlands J.T. Gourse R.L. EMBO J. 1990; 9: 3733-3742Crossref PubMed Scopus (288) Google Scholar), to yield plasmids pYHF1, pYHF2, and pYHF3, respectively. The supercoiled plasmids used as DNA templates for these assays were purified with the Midi Kit from Qiagen. The RNAPs used in these experiments were Eς70 purified from E. coli or a Rhodobacter sphaeroides RNAP preparation enriched for the Eς70 homolog (28Karls R. Donohue T.J. J. Bacteriol. 1993; 175: 7629-7638Crossref PubMed Google Scholar). Standard multiple-round transcription assays (13Ross W. Gosink K.K. Salomon J. Igarashi K. Zou C. Ishihama A. Severinov K. Gourse R.L. Science. 1993; 262: 1407-1413Crossref PubMed Scopus (622) Google Scholar) were modified as described below to accommodate the requirement of CooA for an anoxic environment to bind CO (7Shelver D. Kerby R.L. He Y. Roberts G.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11216-11220Crossref PubMed Scopus (192) Google Scholar). The sealed tubes containing 25-μl reactions (0.2 nm supercoiled plasmid, 3.5 nm RNAP, 40 nm CooA dimer, and a buffer (30 mm KCl, 40 mm Tris acetate, pH 7.9, 10 mmMgCl2, 1 mm dithiothreitol, 100 mg/ml bovine serum albumin, 200 mm ATP, 200 mm CTP, 200 mm GTP)) were degassed and filled with argon in the head space. After the addition of dithionite to 1.7 mm to scavenge any free oxygen, CO was added, and the reactions were incubated at room temperature for 15 min. This step served to activate CooA and allow the activated CooA to interact with the promoter and RNAP to form a predicted 21-nucleotide transcript by incorporating ATP, GTP, and CTP. The reactions were then exposed to air, and 10 mm UTP and 5 μCi of [32P]UTP (DuPont) were added to extend the mRNA at room temperature for 20 min. Reactions were terminated and electrophoresed as described (29Leirmo S. Gourse R.L. J. Mol. Biol. 1991; 220: 550-568Crossref Scopus (74) Google Scholar). The signal intensities of transcripts were quantified using a PhosphorImager (Molecular Dynamics) and ImageQuant software. A DNA fragment containing the PcooF sequence from position −90 to +70 was polymerase chain reaction-amplified with an unlabeled bottom strand primer and a top strand primer labeled with [γ-32P]ATP and polynucleotide kinase. The amplified fragment was purified by polyacrylamide gel electrophoresis, followed by an Elutip Minicolumn (Schleicher & Schuell). The labeled fragment was incubated with CooA, or RNAP, or both in the presence of CO and under the stringent anoxic conditions described previously (2He Y. Shelver D. Kerby R.L. Roberts G.P. J. Biol. Chem. 1996; 271: 120-123Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), except that 40 nm pure CooA and 3.5 nm RNAP were used in a 20-μl binding reaction. The reactions were treated with 2 units/ml RQ RNase-free DNase I (Promega) for 30 s. DNase I cleavage products were separated on a 6% (w/v) polyacrylamide-urea gel. Neither heparin nor any nucleotides were added into the reactions. Strains with a PcooF-lacZ reporter were grown aerobically in LB medium containing 100 mg/ml ampicillin and 35 mg/ml chloramphenicol for 12 h to reach stationary phase. In stoppered test tubes, 20-μl cultures were diluted into 2 ml of LB medium supplemented with 20 mm glucose and the same antibiotics. The air in the head space was replaced by argon and 2% CO, and the cultures were grown anaerobically to an A 600 of approximately 0.45. β-Galactosidase activity was determined according to Miller (30Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992Google Scholar). N-terminal His-tagged wild-type and mutant α subunits were overexpressed from plasmid pHTT7f1-NHα (31Tang H. Severinov K. Goldfarb A. Ebright R.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4902-4906Crossref PubMed Scopus (111) Google Scholar) or derivatives constructed by gene replacement of the EcoRI-BamHI fragment with fragments encoding the desired alanine substitutions (14Gaal T. Ross W. Blatter E. Tang H. Jia X. Krishnan V.V. Assa-Munt N. Ebright R.H. Gourse R.L. Genes Dev. 1996; 10: 16-26Crossref PubMed Scopus (170) Google Scholar). Purification of α subunits by Ni2+ affinity chromatography was performed as described in Tang et al. (31Tang H. Severinov K. Goldfarb A. Ebright R.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4902-4906Crossref PubMed Scopus (111) Google Scholar). Preparation of inclusion bodies of β, β′, and ς70 from strains XL1-Blue (pMKSe2), BL21 DE3 (pT7β′), and BL21 DE3 (pLHN12 α), respectively, and reconstitution of RNAP were carried out as described (31Tang H. Severinov K. Goldfarb A. Ebright R.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4902-4906Crossref PubMed Scopus (111) Google Scholar). CooA was purified from an overproducing strain of R. rubrum (UQ459) by the method of Shelver et al. (7Shelver D. Kerby R.L. He Y. Roberts G.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11216-11220Crossref PubMed Scopus (192) Google Scholar). CooA has been shown to be necessary for the CO-dependent expression of the two coo operons of R. rubrum (1Shelver D. Kerby R.L. He Y. Roberts G.P. J. Bacteriol. 1995; 177: 2157-2163Crossref PubMed Google Scholar, 2He Y. Shelver D. Kerby R.L. Roberts G.P. J. Biol. Chem. 1996; 271: 120-123Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), and purified CooA binds DNA in a CO-dependent manner (7Shelver D. Kerby R.L. He Y. Roberts G.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11216-11220Crossref PubMed Scopus (192) Google Scholar). These data are consistent with the hypothesis that CooA is both necessary and sufficient for sensing and activating transcription in response to CO, but they are not conclusive. We therefore examined the requirement for CooA in two heterologous systems. We chose R. sphaeroides because it is related to R. rubrum, yet does not appear to have thecoo system, as judged by the absence of CO dehydrogenase activity and a failure to hybridize to probes of the coogenes (2He Y. Shelver D. Kerby R.L. Roberts G.P. J. Biol. Chem. 1996; 271: 120-123Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In this organism, a pRK404-based plasmid carryingcooFSCTJ with its normal promoter failed to produce detectable CO dehydrogenase activity (the product of cooS) in response to CO. However, when cooA was added to the plasmid in its normal position at the 3′ end of the cooFSCTJoperon, exposure of the R. sphaeroides strain carrying this plasmid to CO produced easily detectable CO dehydrogenase activity (2He Y. Shelver D. Kerby R.L. Roberts G.P. J. Biol. Chem. 1996; 271: 120-123Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). We then examined CO- and CooA-dependent transcription inE. coli, an organism that is less related to R. rubrum and also lacks any evidence of a coo system. For this test, a reporter system was constructed that contained a PcooF-lacZ fusion in the chromosome and a plasmid overexpressing CooA. In this system, we detected a substantial increase of β-galactosidase activity upon CO induction (200 Miller units in the presence of CO and 1.6 units in its absence), suggesting that CooA is sufficient for activating the transcription of PcooF inE. coli. Similar results with CooA reporters in E. coli have recently been reported by others (9Aono S. Ohkubo K. Matsuo T. Nakajima H. J. Biol. Chem. 1998; 273: 25757-25764Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). These results establish that CooA is necessary for the transcriptional response to CO and that it is able to associate productively in vivo with RNAPs from both E. coli and R. sphaeroides. The above results indicate that CooA is necessary, but only in vitro analysis can establish whether it is sufficient for CO-dependent transcriptional activation or whether additional factors are required for this activation. The ability of CooA to activate PcooF was studied by monitoring RNA synthesis in a purified system containing only DNA, RNAP, CooA, nucleotide triphosphates, and the proper buffer. To investigate the nature of CooA-mediated activation at the PcooFpromoter, we modified the standard in vitro transcription assay, because CooA is only able to bind CO when reduced. The modified assay, detailed under “Experimental Procedures,” was kept anoxic in the presence of CO to support activation by CooA until the formation of a 21-nucleotide transcript from PcooF. At this point, the reaction was exposed to air and extended aerobically for technical convenience. When the reactions were maintained strictly anoxically throughout the entire experiment, a quantitatively similar result was obtained, indicating that the in vitro transcription assay conditions we employed were sufficient for maximal CooA activity (data not shown). The in vitro transcription assays were performed with a supercoiled DNA template (pYHF1) containing the PcooF and extending −250 base pairs upstream and +70 base pairs downstream of transcription start site. The reactions were carried out in the presence or absence of CO. As shown in lanes 2 and4 of Fig. 1, CO-dependent transcripts were detected using RNAP from bothR. sphaeroides and E. coli. The observed size of the transcript from PcooF correlated well with the predicted size of 240 nucleotides. In the absence of CO, no transcripts from PcooF were seen, whereas transcription of the control (RNA-1) was not affected by CO (Fig. 1, lanes 1 and3). These results demonstrate that CooA is sufficient for CO-dependent transcriptional activation and that no other factors are required. The results with the Eς70 fromE. coli indicate that PcooF can be recognized by ς70 when activated by CooA. Because the α-CTD makes specific DNA contacts at some promoters and specific protein contacts with a number of transcription activators including the CooA homolog CRP (13Ross W. Gosink K.K. Salomon J. Igarashi K. Zou C. Ishihama A. Severinov K. Gourse R.L. Science. 1993; 262: 1407-1413Crossref PubMed Scopus (622) Google Scholar, 20Busby S. Ebright R.H. Mol. Microbiol. 1997; 23: 853-859Crossref PubMed Scopus (159) Google Scholar), we wished to test whether there were similar contacts between α-CTD and either CooA or PcooF. We first examined whether the C-terminal domain of the RNAP α subunit is required for transcription activation of PcooF by CooA. To address this question, we assayed the ability of a reconstituted Eς70 containing a truncation of the C-terminal domain of α to direct transcription from CooA-dependent promoter PcooF in vitro. Use of E. coli RNAP with the α-CTD truncation resulted in a dramatic reduction of transcription from PcooF (Fig. 1,lanes 5 and 6). The enzyme activities of wild-type and mutant RNAPs were similar as demonstrated by transcription from the RNA-1 promoter. With a longer exposure of the x-ray film, we were able to detect a low level of a CO-dependent transcript from PcooF using RNAP with α-235 (data not shown). Because the α-CTD is known to make contacts with activators and UP elements, the ineffectiveness of the α-CTD-truncated RNAP for PcooF transcription suggests that the α truncation disrupts the binding of α to CooA, to a UP element, or to both. To analyze potential protein-protein interaction between CooA and RNAP and to specifically address interactions between α-CTD and CooA, we performed DNase I footprinting experiments using wild-type RNAP or the α-CTD-truncated RNAP in the presence or absence of CooA. As shown in Fig.2, lanes 2–4, CooA alone protected PcooF on the top strand from positions −55 to −31 relative to the start site of transcription, in agreement with our previous observations (2He Y. Shelver D. Kerby R.L. Roberts G.P. J. Biol. Chem. 1996; 271: 120-123Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 7Shelver D. Kerby R.L. He Y. Roberts G.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11216-11220Crossref PubMed Scopus (192) Google Scholar). In contrast, neither the wild-type nor the α-CTD-truncated RNAPs alone protected PcooF from DNase I (lanes 5 and 7). This result indicates that RNAP does not form a stable complex at PcooF in the absence of CooA. When both CooA and wild-type RNAP were incubated together with the DNA fragment, the protected region extended from the CooA-binding site in both directions, upstream to −68 and downstream to at least −1 (lanes 8 and 9). A similar result was obtained when R. sphaeroides RNAP was used, indicating that the RNAPs from these two different organisms function similarly in interacting with CooA at PcooF (lanes 10–12). In contrast to the results obtained with the wild-type RNAP, the α-CTD-truncatedE. coli RNAP showed no evidence of forming a stable complex even in the presence of CooA (lane 6). This suggests that CooA activates transcription by enhancing the initial and stable binding of RNAP to PcooF through direct protein-protein contact with the α-CTD. As noted above, the presence of CooA and wild-type RNAP, but not the α-CTD-truncated variants, caused DNase I protection to extend upstream and downstream of that region protected by CooA alone. The region upstream of the CooA-binding site is A + T-rich (Fig.3) relative to most of the R. rubrum genome, reminiscent of the A + T-richness of UP elements inE. coli promoters that contact the α-CTD to increase transcription (13Ross W. Gosink K.K. Salomon J. Igarashi K. Zou C. Ishihama A. Severinov K. Gourse R.L. Science. 1993; 262: 1407-1413Crossref PubMed Scopus (622) Google Scholar, 38). To test whether α interacts with this region in a sequence-specific manner, we created PcooF constructs differing only in the extent of that region. PcooF sequences from positions −90 to +70 and −60 to +70 were cloned into the transcription assay vector pRLG770, resulting in the constructs pYHF2 and pYHF3, respectively. These plasmids were tested for CooA-dependent in vitro transcription activity with wild-type RNAP. The level of transcripts from those constructs was quantitatively compared with that of the construct pYHF1 containing the PcooF sequences from −250 to +70. As shown in Fig.4, these three templates yielded similar amounts of CooA-dependent transcripts, indicating that the specific sequences upstream of position −60 in PcooF do not contribute significantly to promoter activity. This also suggests that the upstream A + T-rich sequence of PcooF does not act as a UP element. Although we cannot exclude the possibility that some portion of the protection upstream of the CooA-binding site is due to a conformational change in CooA induced by the presence of RNAP, the interaction of α-CTD with DNA upstream of CooA is consistent with the sequence-nonspecific interactions between DNA and α-CTD observed at class II CRP-dependent promoters (23Savery N.J. Lloyd G.S. Kainz M. Gaal T. Ross W. Ebright R.H. Gourse R.L. Busby S. EMBO J. 1998; 17: 3439-3447Crossref PubMed Scopus (118) Google Scholar).Figure 4In vitro transcription analysis of PcooF deletions. The transcription reactions were performed in the same manner as described in Fig. 1. All of the reactions contained wild-type RNAP from E. coli, CooA, and CO. The supercoiled DNA templates used in the assay differ only in the extent of the upstream sequence of PcooF. Lane 1, pYHF3 containing PcooF sequences from −60 to +70; lane 2, pYHF2 containing PcooF sequences from −90 to +70;lane 3, pYHF1 containing PcooF sequences from −250 to +70. The positions of transcripts initiated at the PcooF and the RNA-1 promoter are indicated by arrows.View Large Image Figure ViewerDownload (PPT) We wished to determine whether CooA made similar contacts with the α-CTD as seen with its homolog, CRP, and also to test whether the extended DNase I protection upstream of CooA reflected the direct contact of α-CTD with DNA. To address these questions, we measured CooA-activated transcription using a CooA reporter system containing a PcooF-lacZ fusion integrated into the E. coli chromosome, a plasmid expressing CooA, and a library of plasmids encoding single alanine substitutions throughout the entire α-CTD (23Savery N.J. Lloyd G.S. Kainz M. Gaal T. Ross W. Ebright R.H. Gourse R.L. Busby S. EMBO J. 1998; 17: 3439-3447Crossref PubMed Scopus (118) Google Scholar). The effects of the α-CTD mutants in this screen were small and somewhat variable (data not shown), but they did identify a few potential mutants that were then assayed for their effects on CooA-dependent transcription in vitro. We tested reconstituted RNAPs in vitro containing single alanine substitutions for Thr285, Val287, Glu288, and Arg317 (the patch on α that interacts with CRP at class II CRP-dependent promoters (23Savery N.J. Lloyd G.S. Kainz M. Gaal T. Ross W. Ebright R.H. Gourse R.L. Busby S. EMBO J. 1998; 17: 3439-3447Crossref PubMed Scopus (118) Google Scholar)), Arg265 (the residue most important for interaction of α with DNA (14Gaal T. Ross W. Blatter E. Tang H. Jia X. Krishnan V.V. Assa-Munt N. Ebright R.H. Gourse R.L. Genes Dev. 1996; 10: 16-26Crossref PubMed Scopus (170) Google Scholar)), and a few additional mutants suggested by the in vivo screen, including Val306, Leu307, and Ser313. The results of the in vitro transcription analysis with a subset of the variant RNAPs are shown in Fig.5. The activity of each RNAP preparation was normalized to the transcription from the RNA-1 promoter. The R265A, L307A, and V287A RNAPs were defective in CooA-dependent transcription of PcooF, providing 13, 34, and 37% of wild-type RNAP activity, respectively (Fig. 5, lanes 2, 3, and 7). Because R265A (14Gaal T. Ross W. Blatter E. Tang H. Jia X. Krishnan V.V. Assa-Munt N. Ebright R.H. Gourse R.L. Genes Dev. 1996; 10: 16-26Crossref PubMed Scopus (170) Google Scholar) and L307A 2S. Bales, M. Burgess, S. Aiyar, and R. L. Gourse, unpublished data. affected UP element-dependent transcription and RNAP extended the DNase I protection upstream of the CooA-binding site, this strongly suggested that α-CTD makes contacts with DNA upstream of CooA and that these contacts are important for CooA-meditated transcription activation. V287A was also defective in CooA-dependent transcription, although the other α-CTD variants important for class II CRP-dependent transcription (T285A, E288A, and R317A) had little or no effect. These results suggest that the CooA contact site in the α-CTD of RNAP shares some determinants of, but is not identical to, the site for CRP contact at class II-type promoters. RNAPs containing α mutants V306A and S313A, which were also suggested by the in vivo screen, were not defective in CooA-dependent transcription in vitro (data not shown). Many transcription activators directly contact the α subunit of RNAP (16Ebright R.H. Busby S. Curr. Opin. Genet. Dev. 1995; 5: 197-203Crossref PubMed Scopus (136) Google Scholar, 17Ishihama A. Mol. Microbiol. 1992; 6: 3283-3288Crossref PubMed Scopus (138) Google Scholar). In this study, we determined that CooA is sufficient for directing RNAP to initiate transcription of PcooF and that CooA-activated transcription of PcooF requires the C-terminal domain of the RNAP α subunit. Consistent with these observations, CooA facilitates the binding of wild-type RNAP to PcooF but not that of α-CTD-truncated RNAP. These observations suggest that direct protein-protein contact between CooA and the α-CTD of RNAP plays an essential role in transcription activation of PcooF. We suspect that the α-CTD/CooA interaction plays a role similar to that between AR1 of CRP and α-CTD at class II CRP-dependent promoters. In class II CRP-dependent promoters, α-CTD makes nonspecific contacts with the DNA segment immediately upstream of the CRP site (16Ebright R.H. Busby S. Curr. Opin. Genet. Dev. 1995; 5: 197-203Crossref PubMed Scopus (136) Google Scholar, 23Savery N.J. Lloyd G.S. Kainz M. Gaal T. Ross W. Ebright R.H. Gourse R.L. Busby S. EMBO J. 1998; 17: 3439-3447Crossref PubMed Scopus (118) Google Scholar). In this study, we found that wild-type RNAP, but not RNAP with the α-CTD truncation, extends DNase I protection upstream of that protected by CooA alone. Furthermore, we determined that α R265A and L307A, which decrease UP element-dependent transcription, also affect PcooF activity. Because the specific DNA sequence upstream of the CooA-binding site is not critical for PcooF activity, we propose that α-CTD makes nonspecific contacts with the DNA immediately upstream of the CooA site and that this protein-DNA interaction is important for CooA-activated transcription of PcooF. Val287 is also important for CooA-activated transcription, although the other tested α residues that contact CRP at class II promoters are not critical for CooA-dependent transcription of PcooF. Therefore, a different set of side chains, but probably the same region of α-CTD, might be important for activation by CRP and CooA. This hypothesis is consistent with the low similarity between CRP and CooA in activating region 1 (22Zhou Y. Pendergrast P.S. Bell A. Williams R. Busby S. Ebright R. EMBO J. 1994; 13: 4549-4557Crossref PubMed Scopus (81) Google Scholar). In the DNase I footprinting analysis, RNAP does not completely protect the DNA downstream of PcooF +1, even in the presence of CooA and in the absence of heparin (Fig. 2). In addition, the majority of the CooA-RNAP-PcooF complexes detected in gel-shift assays are heparin-sensitive (data not shown). This partial downstream protection and the heparin sensitivity suggest that the detected ternary complex (CooA-RNAP-PcooF) may be a mixed population of closed and open complexes. Although most CRP-dependent promoters, such aslacP1, melT, galP1, form stable open complexes with RNAP in the presence of CRP (32Tagami H. Aiba H. Nucleic Acids Res. 1995; 23: 599-605Crossref PubMed Scopus (41) Google Scholar, 33Eichenberger P. Dethiollaz Z. Fujiti N. Ishihama A. Geiselmann J. Biochemistry. 1996; 35: 15302-15312Crossref PubMed Scopus (15) Google Scholar, 34Herbert M. Kolb A. Buc H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2807-2811Crossref PubMed Scopus (40) Google Scholar, 35Tagami H. Aiba H. EMBO J. 1998; 17: 1759-1768Crossref PubMed Scopus (41) Google Scholar), other promoters, such as rrnB P1 even in the presence of its activator protein Fis (36Bokal A.J. Ross W. Gaal T. Johnson R.C. Gourse R.L. EMBO J. 1997; 16: 154-162Crossref PubMed Scopus (74) Google Scholar), form an unstable open complex with RNAP (37Gaal T. Bartlett M.S. Ross W. Turnbough C.L. Gourse R.L. Science. 1997; 278: 2092-2097Crossref PubMed Scopus (290) Google Scholar). Because basal level transcription of PcooF in the absence of active CooA is not detectable, it is formally possible that the deletion of α-CTD affects basal level transcription of PcooFand not its activation by CooA. However, the requirement for at least one residue in α-CTD (Val287) that has no effect on DNA binding (23Savery N.J. Lloyd G.S. Kainz M. Gaal T. Ross W. Ebright R.H. Gourse R.L. Busby S. EMBO J. 1998; 17: 3439-3447Crossref PubMed Scopus (118) Google Scholar) suggests that the α-CTD requirement involves, at least in part, a protein-protein interaction between CooA and α-CTD. We thank Daniel Shelver for providing pure CooA and Marcin Filutowicz for suggestions and for reading the manuscript." @default.
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