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• W2063452009 abstract "The “σ” subunit of prokaryotic RNA polymerase allows gene-specific transcription initiation. Two σ families have been identified, σ70 and σ54, which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the σ70-type factors have been well characterized structurally by x-ray crystallography, no high resolution structural information is available for the σ54-type factors. Here we present the NMR-derived structure of the C-terminal domain of σ54 from Aquifex aeolicus. This domain (Thr-323 to Gly-389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three-helix bundle, which is surprisingly similar to domains of the σ70-type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. The homology of this structure with other DNA-binding proteins, combined with previous biochemical data, suggests how the C-terminal domain of σ54 binds to DNA. The “σ” subunit of prokaryotic RNA polymerase allows gene-specific transcription initiation. Two σ families have been identified, σ70 and σ54, which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the σ70-type factors have been well characterized structurally by x-ray crystallography, no high resolution structural information is available for the σ54-type factors. Here we present the NMR-derived structure of the C-terminal domain of σ54 from Aquifex aeolicus. This domain (Thr-323 to Gly-389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three-helix bundle, which is surprisingly similar to domains of the σ70-type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. The homology of this structure with other DNA-binding proteins, combined with previous biochemical data, suggests how the C-terminal domain of σ54 binds to DNA. Transcription, the synthesis of RNA from double-stranded DNA, is a fundamental process in all forms of life. The primary protein complex catalyzing transcription is composed of five subunits, α2β′βω, called “core RNA polymerase” (RNAP). 2The abbreviations used are: RNAPRNA polymeraseNOEnuclear Overhauser effectNOESYNOE spectroscopyTOCSYtotal correlation spectroscopyHSQChetero-nuclear single quantum coherence spectroscopyPDBProtein Data Bankr.m.s.d.root mean square deviation.2The abbreviations used are: RNAPRNA polymeraseNOEnuclear Overhauser effectNOESYNOE spectroscopyTOCSYtotal correlation spectroscopyHSQChetero-nuclear single quantum coherence spectroscopyPDBProtein Data Bankr.m.s.d.root mean square deviation. Core RNAP is fully competent to synthesize RNA from DNA. However, the initiation of transcription at specific DNA sequences requires additional protein(s). In bacteria, these additional proteins are called the “σ factors” (1Burgess R.R. Travers A.A. Dunn J.J. Bautz E.K. Nature. 1969; 221: 43-46Crossref PubMed Scopus (631) Google Scholar, 2Hirschman J. Wong P.K. Sei K. Keener J. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7525-7529Crossref PubMed Scopus (185) Google Scholar). The σ factor facilitates transcription at specific DNA sequences by 1) binding to the core RNAP to form the σ-RNAP holoenzyme, 2) recognizing and binding to a specific DNA sequence next to the transcription start site, called the promoter element, and 3) opening the double-stranded DNA to initiate transcription. RNA polymerase nuclear Overhauser effect NOE spectroscopy total correlation spectroscopy hetero-nuclear single quantum coherence spectroscopy Protein Data Bank root mean square deviation. RNA polymerase nuclear Overhauser effect NOE spectroscopy total correlation spectroscopy hetero-nuclear single quantum coherence spectroscopy Protein Data Bank root mean square deviation. Based on protein sequence homology, σ factors can be grouped into two classes, σ70 and σ54, which were named by the molecular weights of the first members identified (reviewed in Ref. 3Wosten M.M. FEMS Microbiol. Rev. 1998; 22: 127-150Crossref PubMed Scopus (305) Google Scholar). The σ70-type factors are the most abundant in bacteria (reviewed in Refs. 4Lonetto M. Gribskov M. Gross C.A. J. Bacteriol. 1992; 174: 3843-3849Crossref PubMed Scopus (729) Google Scholar, 5Paget M.S. Helmann J.D. Genome Biol. 2003; 4: 203Crossref PubMed Scopus (345) Google Scholar). They include the primary σ factors, such as σ70 from Escherichia coli and σA from Gram-positive bacteria, which regulate transcription for most genes required for normal exponential growth. Other members of the σ70 family (σ28, σ32, etc.) regulate the transcription of more specialized genes that are required to respond to environmental changes. For example, σ28 from Salmonella typhimurium controls expression of genes required for flagellar assembly (6Chilcott G.S. Hughes K.T. Microbiol. Mol. Biol. Rev. 2000; 64: 694-708Crossref PubMed Scopus (485) Google Scholar). The σ54-type factor, which is also called σN and encoded by the rpoN gene, has no detectable sequence homology to σ70-type factors. Its occurrence is widespread among bacteria, but there is usually only one σ54 gene present in a particular organism (7Buck M. Gallegos M.T. Studholme D.J. Guo Y. Gralla J.D. J. Bacteriol. 2000; 182: 4129-4136Crossref PubMed Scopus (348) Google Scholar). σ54 regulates gene transcription for proteins required for many important cellular functions, such as nitrogen metabolism, development, phage shock responses, and pathogenicity. For example, σ54 has been shown to be required for mammalian infection by Borrelia burgdorferi, the agent of Lyme disease (8Fisher M.A. Grimm D. Henion A.K. Elias A.F. Stewart P.E. Rosa P.A. Gherardini F.C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5162-5167Crossref PubMed Scopus (180) Google Scholar). The most striking difference between σ54 and σ70 factors is how they regulate transcription initiation. Once σ70-RNAP holoenzyme binds to the promoter of a gene (at a conserved sequence -10 and -35 base pairs upstream of the transcription start site), it can open DNA and transcription can begin spontaneously. In contrast, when σ54-RNAP holoenzyme binds to a promoter (at a consensus sequence -12 and -24 base pairs upstream of the transcription start site), it remains in the closed, inactive state. To open the double-stranded DNA and initiate transcription, σ54-RNAP holoenzyme requires interaction with an activator protein (reviewed in Ref. 9Xu H. Hoover T.R. Curr. Opin. Microbiol. 2001; 4: 138-144Crossref PubMed Scopus (97) Google Scholar), which binds ∼150 base pairs upstream of the promoter (10Wedel A. Weiss D.S. Popham D. Droge P. Kustu S. Science. 1990; 248: 486-490Crossref PubMed Scopus (151) Google Scholar). Once stimulated by a physiological signal, such as phosphorylation or ligand binding, the activator uses the energy of ATP hydrolysis (via a conserved AAA+-ATPase domain) to remodel the σ54-RNAP promoter complex into an active conformation that is capable of initiating transcription (7Buck M. Gallegos M.T. Studholme D.J. Guo Y. Gralla J.D. J. Bacteriol. 2000; 182: 4129-4136Crossref PubMed Scopus (348) Google Scholar). The σ70-type factors have been well characterized structurally (11Malhotra A. Severinova E. Darst S.A. Cell. 1996; 87: 127-136Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 12Campbell E.A. Tupy J.L. Gruber T.M. Wang S. Sharp M.M. Gross C.A. Darst S.A. Mol. Cell. 2003; 11: 1067-1078Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 13Jain D. Nickels B.E. Sun L. Hochschild A. Darst S.A. Mol. Cell. 2004; 13: 45-53Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 14Sorenson M.K. Ray S.S. Darst S.A. Mol. Cell. 2004; 14: 127-138Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). X-ray crystal structures of individual domains of σ70 and σ70-RNAP holoenzyme show that σ70-type factors are composed of four helical domains (σ1, σ2, σ3, σ4) connected by flexible linkers (15Campbell E.A. Muzzin O. Chlenov M. Sun J.L. Olson C.A. Weinman O. Trester- Zedlitz M.L. Darst S.A. Mol. Cell. 2002; 9: 527-539Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 16Murakami K.S. Masuda S. Darst S.A. Science. 2002; 296: 1280-1284Crossref PubMed Scopus (441) Google Scholar). These four domains are also functionally distinct: σ1 is involved in regulating the kinetics of transcription initiation; σ2 binds to the -10 promoter element and is essential for melting the DNA; σ3 stabilizes the open complex formation by binding the extended -10 element; and σ4 binds specifically to the -35 promoter element (reviewed in Ref. 17Murakami K.S. Darst S.A. Curr. Opin. Struct. Biol. 2003; 13: 31-39Crossref PubMed Scopus (380) Google Scholar). In contrast to σ70, σ54 is divided into three regions based on function (18Wong C. Tintut Y. Gralla J.D. J. Mol. Biol. 1994; 236: 81-90Crossref PubMed Scopus (56) Google Scholar, 19Cannon W.V. Chaney M.K. Wang X. Buck M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5006-5011Crossref PubMed Scopus (34) Google Scholar, 20Bordes P. Wigneshweraraj S.R. Schumacher J. Zhang X. Chaney M. Buck M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2278-2283Crossref PubMed Scopus (77) Google Scholar). Region 1 (E. coli-(1-55)) interacts with the upstream activator protein to control promoter melting; region 2 (E. coli-(70-304)) contains the minimal RNAP-binding domain (E. coli-(70-180) and a part that enhances DNA binding affinity (E. coli-(180-304)); region 3 (E. coli-(329-477)) recognizes and binds the consensus promoter elements. This C-terminal part of the protein contains a region that cross-links to DNA (E. coli-(329-346)), a predicted helix-turn-helix motif (E. coli-(366-386); Fig. 1), and a highly conserved sequence (E. coli-(454-463); Fig. 1) called the RpoN box. The RpoN box (ARRTVAKYRE) is the signature sequence for σ54 proteins and is critical for DNA binding (21Taylor M. Butler R. Chambers S. Casimiro M. Badii F. Merrick M. Mol. Microbiol. 1996; 22: 1045-1054Crossref PubMed Scopus (44) Google Scholar). Although information about the overall shape of σ54 has come from scattering and electron microscopy data (22Svergun D.I. Malfois M. Koch M.H. Wigneshweraraj S.R. Buck M. J. Biol. Chem. 2000; 275: 4210-4214Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 23Rappas M. Schumacher J. Beuron F. Niwa H. Bordes P. Wigneshweraraj S. Keetch C.A. Robinson C.V. Buck M. Zhang X. Science. 2005; 307: 1972-1975Crossref PubMed Scopus (137) Google Scholar), no high resolution structural information is available for σ54. The lack of sequence homology between σ54 and σ70 prevents homology modeling of σ54 domains based on the structures of σ70 domains. Therefore, to understand the mechanism of σ54-dependent transcription initiation, high resolution structures of domains are required. Here we present the NMR-derived structure of a C-terminal fragment of σ54 (residues Thr-323 to Gly-389) from the hyperthermophilic bacterium, Aquifex aeolicus. This domain, which includes the signature RpoN box motif, consists of a poorly structured N-terminal tail followed by an unpredicted helix-turn-helix motif. Surprisingly, this domain is structurally similar to the σ3 and σ4 domains of σ70-type proteins, despite their low sequence homology. Structural homology with other DNA-binding proteins suggests how this protein may bind the -24 promoter element. Because of the high sequence conservation of this region among σ54 proteins (Fig. 1), it is likely that this domain has a similar fold in all species, including the most highly studied σ54 proteins from E. coli and Klebsiella pneumonia. Sequence Analyses—σ54 protein sequences from a variety of species were downloaded from the National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov/) and then aligned using the ClustalW (24Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (54882) Google Scholar) web server at the European Bioinformatics Institute (www.ebi.ac.uk/clustalw/). Secondary structure prediction for the A. aeolicus σ54 protein sequence was performed using the neural network Jnet (25Cuff J.A. Barton G.J. Proteins. 2000; 40: 502-511Crossref PubMed Scopus (655) Google Scholar) via the Jpred web server (www.compbio.dundee.ac.uk/~www-jpred/) (26Cuff J.A. Clamp M.E. Siddiqui A.S. Finlay M. Barton G.J. Bioinformatics. 1998; 14: 892-893Crossref PubMed Scopus (907) Google Scholar). Plasmid Construction—Six unique constructs of the C-terminal region of σ54 from A. aeolicus were cloned by PCR with primers (Operon) containing NdeI and BamHI restriction sites at the 5′ and 3′-ends, respectively. The fragments were amplified from a plasmid containing the full-length σ54 gene (courteously provided by Gongyi Zhang from the National Jewish Medical Center, Denver, CO). The amplified fragments were digested with NdeI and BamHI, purified by gel electrophoresis, and ligated into the pET21a expression vector. Protein Preparation—σ54 C protein was expressed using E. coli BL21 (DE3) with Rosetta.pLysS plasmid. Cells were first grown at 37 °C in two liters of Luria-Bertani medium. At A600 nm ∼0.6, cells were exchanged (27Marley J. Lu M. Bracken C. J. Biomol. NMR. 2001; 20: 71-75Crossref PubMed Scopus (601) Google Scholar) into one liter of M9 minimal medium containing 1 g of 15N-NH4Cl and/or 2 g of 13C6-glucose (Cambridge Isotope Laboratory). Four hours after induction with isopropyl-1-thio-β-d-galactopyranoside, cells were harvested by centrifugation and resuspended into 50 mm Hepes (pH 6.9), 50 mm NaCl, 1 mm EDTA, and 0.1 mm phenylmethylsulfonyl fluoride. σ54 C protein was purified by ion exchange chromatography (heparin; Amersham Biosciences) and by size exclusion chromatography (Superdex 75; Amersham Biosciences). For NMR spectroscopy, σ54C protein was concentrated to 0.8 mm in 50 mm Hepes (pH 6.9), 250 mm NaCl, 1 mm EDTA, 10% 2H2O, and 1× Complete Protease Inhibitor mixture (Roche Applied Science). Resonance Assignments—NMR data were collected at 298 K on a Bruker DRX 600 MHz. All data were processed with NMRPipe (28Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11273) Google Scholar) and analyzed with NMRView (29Johnson B.A. Blevins B.A. J. Biomol. NMR. 1994; : 4Google Scholar). Small amounts of proteolysis were detected by new peaks in the 15N-HSQC spectrum after 5 days at 298 K. Therefore, a fresh protein sample was used for each three-dimensional spectrum. Backbone assignments were obtained using the three-dimensional triple resonance experiments HNCA, CBCA(CO)NH, and 15N-NOESY-HSQC. Side chain assignments were obtained from the three-dimensional experiments 15N-TOCSY-HSQC, C(CO)NH, and HCCH-TOCSY, as well as the two-dimensional experiment proton DQF-COSY for the aromatic proton assignments. Distance Restraints—Backbone dihedral restraints were obtained from the backbone chemical shifts (HN, HA, CA, and CB) using the program TALOS (30Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2727) Google Scholar). 3JHNHA-coupling constants were obtained from an HNHA spectrum (31Kuboniwa H. Grzesiek S. Delaglio F. Bax A. J. Biomol. NMR. 1994; 4: 871-878Crossref PubMed Scopus (334) Google Scholar). Nuclear Overhauser effect (NOE) distance constraints were derived by manual assignment of cross-peaks in a three-dimensional 15N-NOESY-HSQC and 13C-NOESY-HSQC, both with 100-ms mixing times. Hydrogen bond restraints were defined from slowly exchanging amide protons. The protein was exchanged into 95% D2O using an Amicon Ultra centrifugal concentrator (5-kDa cutoff; Millipore). Amide protons with strong cross-peak intensities in the 15N-HSQC after 3 h at 298 K were identified as significantly protected. Stereo-specific assignments of the methyl groups of valine and leucine residues were obtained from a 13C-HMQC spectrum of a 10% 13C-labeled protein sample as described in Ref. 32Neri D. Szyperski T. Otting G. Senn H. Wuthrich K. Biochemistry. 1989; 28: 7510-7516Crossref PubMed Scopus (563) Google Scholar. Only residues with totally unambiguous methyl assignments were used for stereo-specific assignments. Structure Calculations—Structure calculations were performed with the program DYANA (33Guntert P. Mumenthaler C. Wuthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2534) Google Scholar). Initial structures were calculated using only unambiguous NOESY peak assignments. During the later stages of assignment (backbone r.m.s.d. <1.5 for residues 21-67), the structure with the lowest energy was used to filter possible assignments based on distances. The program MOLMOL (34Koradi R. Billeter M. Wuthrich K. J. Mol. Graph. 1996; 14 (51-55): 29-32Crossref Scopus (6453) Google Scholar) was used to analyze molecules throughout the assignment process. Structure statistics, including PROCHECK analysis (35Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), are summarized in TABLE ONE.TABLE ONEExperimental restraints and structural statistics for the C-terminal domain of σ54Restraints1064Total distance956Intraresidue458Sequential (i-j = 1)201Medium range (2 ≥ I-j ≤5)128Long range (i-j ≥5)169Dihedral ϕ/φ angle restraintsTALOS prediction643JHNHA coupling44Total restraints1064Residual violationsaThe average number of distance and angle violations for each of the 20 final conformers is reported.Number of distance violations ≥0.3 Å2.74 ± 1.42Number of dihedral angle violations ≥5°0 ± 0Number of 3JHNHA coupling violations ≥0.5 Hz0 ± 0Number of van der Waals violations ≥0.2 Å1.1 ± 0.91Average DYANA target function (Å2)2.60 ± 0.33Coordinate precision (Å)br.m.s.d. values are calculated from the mean structure.Backbone atoms in well structured region (residues Gln-340 to Leu-388)0.56 ± 0.07Heavy atoms in well structured region (residues Gln-340 to Leu-388)1.24 ± 0.09Procheck statisticscStructures were not energy minimized.Residues in the most favored region (%)75.8Residues in additional allowed regions (%)20.8Residues in generously allowed regions (%)3.3Residues in disallowed regions (%)0.2a The average number of distance and angle violations for each of the 20 final conformers is reported.b r.m.s.d. values are calculated from the mean structure.c Structures were not energy minimized. Open table in a new tab Miscellaneous—All figures were made with Pymol (www.pymol.org). The sequence alignments were plotted with TexShade (36Beitz E. Bioinformatics. 2000; 16: 135-139Crossref PubMed Scopus (251) Google Scholar). Sequence Analysis and Protein Construct Design—To help identify a subdomain of the σ54 protein suitable for structure determination by NMR, the A. aeolicus σ54 protein sequence was aligned with 38 other σ54 proteins using the program ClustalW (24Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (54882) Google Scholar). This alignment, which includes σ54 proteins from 11 of the most studied species, is shown in Fig. 1. Visual examination of the sequence alignment indicated that the C-terminal end (∼288-398 in A. aeolicus and 360-477 in E. coli) was the most conserved region. This fragment of σ54 has been shown to be sufficient for binding to double-stranded DNA (19Cannon W.V. Chaney M.K. Wang X. Buck M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5006-5011Crossref PubMed Scopus (34) Google Scholar). Using the secondary structure prediction of the A. aeolicus σ54 protein as a guide, six different constructs of this region were cloned and expressed in E. coli. Constructs were created using PCR with three different N-terminal primers and two different C-terminal primers, shown in Fig. 1. Only one of the six fragments expressed in E. coli with high levels of soluble protein. This fragment, shown with ** above the primers in Figs. 1 and 2B, starts at Thr-323, just upstream of a pair of conserved phenylalanines (329, 330) and ends at Gly-389, just downstream of the highly conserved RpoN box motif (377-386; boxed in Fig. 1). For this part of σ54, the A. aeolicus protein sequence is 36% identical to the E. coli protein sequence. Here, we present the solution structure of this 67-residue (7.9 kDa) fragment, which we refer to as “σ54C.” As shown in Fig. 1, this fragment does not include the region predicted to form a helix-turn-helix motif, residues 292-313 in A. aeolicus and 366-386 in E. coli (gray box above the sequence alignment in Fig. 1). Structure Determination—The σ54C fragment gave an excellent 1H-15N HSQC spectrum with highly dispersed cross-peaks (supplemental Fig. S1), indicating that the majority of the protein fragment was stably folded. The 1H-15N HSQC spectrum contained cross-peaks for all residues except for the first three N-terminal residues (Thr-323, Tyr-324, Ser-325) and the single proline (Pro-359). 1H, 15N, and 13C resonance assignments for the σ54C domain were made using standard three-dimensional NMR techniques (37Cavanagh J. Wayne J.F. Palmer A.G. Nicholas J.S. Protein NMR Spectroscopy: Principles and Practice. 1996; (Academic Press, San Diego, CA)Google Scholar). In total, the chemical shifts for 97% of the backbone atoms (N, NH, CA, HA) and 94% of side chain atoms (aliphatic/aromatic C and H) were assigned. Even before we started calculating structures, three properties of the NMR data indicated that the N-terminal portion of σ54C from Leu-326 to Thr-339 was poorly structured and dynamic on a time scale of micro-seconds to seconds. First, in all two- and three-dimensional spectra, diagonal and cross-peak intensities for atoms in this N-terminal region were significantly weaker than for atoms in other parts of the protein. For example, in the 1H-15N HSQC the average relative peak height for residues Leu-326 to Thr-339 is 24% less than the average intensity for residues Gln-340 to Gly-389 (Fig. 2B, last row). Second, chemical shift index (38Spera S. Bax A. J. Am. Chem. Soc. 1991; 113: 5490-5492Crossref Scopus (989) Google Scholar) for this region indicated little secondary structure beyond Leu-338 (Fig. 2B, first two rows). Third, only a small number of inter-residue NOE cross-peaks were present in the three-dimensional 13C-NOESY-HSQC for this region of the protein. Nine hundred fifty-six unique distance restraints (TABLE ONE) were obtained from the three-dimensional 15N-NOESY-HSQC and 13C-NOESY-HSQC spectra. An additional 108 φ/ϕ torsion angle restraints were calculated from HN, HA, CA, and CB chemical shift values (30Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2727) Google Scholar) and from 3JHNHA-coupling constants (31Kuboniwa H. Grzesiek S. Delaglio F. Bax A. J. Biomol. NMR. 1994; 4: 871-878Crossref PubMed Scopus (334) Google Scholar) (TABLE ONE). These 1064 restraints (16 restraints/residue) were used to calculate 150 structures. The 20 structures with the lowest energy were chosen to represent the structure of the C-terminal domain of the σ54 protein from A. aeolicus (Fig. 2A). Structure of the C-terminal Domain of σ54—For the well structured region (Gln-340 to Leu-388) of σ54C, the superposition of the final 20 structures has an r.m.s. deviation from the mean structure of 0.56 ± 0.07 Å for the backbone atoms and 1.24 ± 0.09 Å for all heavy atoms. Overall these structures have good statistics and geometry, as summarized in TABLE ONE. This C-terminal portion of σ54C folds into a compact domain consisting of three α-helices (Fig. 2). Helix 1 (Gln-340 to Asn-353) is followed by a long loop, which starts at the conserved Glu-354 and ends at the conserved Ser-361. Helix 2 (Asp-362 to Leu-369) and helix 3 (Arg-379 to Leu-388), which includes the highly conserved RpoN box sequence, form a helix-turn-helix motif that is commonly found in many DNA-binding proteins (reviewed in Ref. 39Wintjens R. Rooman M. J. Mol. Biol. 1996; 262: 294-313Crossref PubMed Scopus (204) Google Scholar). A color-coded image representing the electrostatic surface of σ54C (residues Gln-340 to Leu-388) shows that the molecule has a striking polarity. It has a positively charged face primarily composed of the loop in the helix-turn-helix motif (loop 2) and the top of helix 3 (Fig. 3C, right). The other side of the molecule is negatively charged, due to negative residues in loop 1 and on helix 1 (supplemental Fig. S2). A search for proteins structurally homologous to σ54C (residues Gln-340 to Leu-388) using the DALI server identified many DNA-binding proteins. Residues 77 to 127 of the human PAX6 homeodomain (PDB accession number 6PAX; Ref. 40Xu H.E. Rould M.A. Xu W. Epstein J.A. Maas R.L. Pabo C.O. Genes Dev. 1999; 13: 1263-1275Crossref PubMed Scopus (234) Google Scholar) had the highest Z score of 5.2 (r.m.s.d 2.1 Å). The N-terminal DNA-binding domain of the leucine-responsive regulatory protein, LrpA, from Pyrococcus furiosus (PDB accession number 1I1G; Ref. 41Leonard P.M. Smits S.H. Sedelnikova S.E. Brinkman A.B. de Vos W.M. van der Oost J. Rice D.W. Rafferty J.B. EMBO J. 2001; 20: 990-997Crossref PubMed Scopus (124) Google Scholar) had the second highest Z score of 4.7 (r.m.s.d. 1.9 Å). The DALI results are summarized in supplemental Table S1 (52Pohl E. Haller J.C. Mijovilovich A. Meyer-Klaucke W. Garman E. Vasil M.L. Mol. Microbiol. 2003; 47: 903-915Crossref PubMed Scopus (258) Google Scholar, 53Mer G. Bochkarev A. Gupta R. Bochkareva E. Frappier L. Ingles C.J. Edwards A.M. Chazin W.J. Cell. 2000; 103: 449-456Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 54Nichols M.D. DeAngelis K. Keck J.L. Berger J.M. EMBO J. 1999; 18: 6177-6188Crossref PubMed Scopus (124) Google Scholar). Interestingly, the σ54C domain has structural homology to both σ3 (regions 3.0-3.1) and σ4 (regions 4.1-4.2) of σ70-type factors. σ3 of σA (residues 279 to 326) from Thermus aquaticus (PDB accession number 1KU2; Ref. 15Campbell E.A. Muzzin O. Chlenov M. Sun J.L. Olson C.A. Weinman O. Trester- Zedlitz M.L. Darst S.A. Mol. Cell. 2002; 9: 527-539Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar) had the tenth highest Z score of 4.1. σ4 of σ28 (residues 186-224) from A. aeolicus (PDB accession number 1RP3; Ref. 14Sorenson M.K. Ray S.S. Darst S.A. Mol. Cell. 2004; 14: 127-138Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) had a lower Z score of 3.8 but aligns slightly better with the σ54C structure (r.m.s.d. 1.9 Å) than the σ3 fragment of σA (r.m.s. deviation of 2.5 Å). Structural alignments with σ4 and σ3 are shown in Fig. 3B, left and right, respectively. As described above, the N-terminal segment from Ser-325 to Thr-339 is poorly structured and probably dynamic on a microsecond-to-second time scale. This type of dynamics causes line broadening, thus making less structural information available for this region. The poor chemical shift dispersion for backbone and side chain atoms (Fig. 2B, top two rows) and the low number of medium- and long-range NOE cross-peaks available made it possible to assign only ten long-range NOE cross-peaks available for this region. These cross-peaks indicate that Ala-335 and Leu-338 interact with the N-terminal end of helix 1 primarily via Leu-343 and with the loop between helices 2 and 3 primarily via Phe-374. For example, the aliphatic hydrogens on Leu-338 interact with the amide and aliphatic hydrogens on Leu-343 and with the aromatic hydrogens on Phe-374. Structural Role for Conserved Residues—The hydrophobic core connecting the three helices is composed of highly conserved residues. For example, Ile-347 in helix 1 makes van der Waal interactions with Ile-365 in helix 2 and Val-381 in helix 3. All three of these residues are isoleucine, leucine, or valine in all 38 σ54 protein sequences examined (11 of these sequences are shown in Fig. 1). Other residues making crucial van der Waals interactions between the helices are Leu-343, Met-344, and Ile-350 in helix 1, Ala-366, Ile-368, and Leu-369 in helix 2, and Tyr-384 and Leu-388 in helix 3. Many of the residues creating the charge on the protein surface are conserved among σ54 proteins. The highly conserved RpoN box (Ala-378 to Glu-386) forms the majority of helix 3, the second helix in the helix-turn-helix motif. This helix is amphipathic, with the hydrophobic face packing against the hydrophobic core described above. The hydrophilic side is surface exposed. This helix is primarily positively charged due to the side chains Arg-378, Arg-379, Lys-383, and Arg-385, but the C-terminal part of the helix has negative charge due to the side chain of Glu-386 (Fig. 3C, right). When fragments of σ70 were crystallized, they were found to be small helical domains (15Campbell E.A. Muzzin O. Chlenov M. Sun J.L. Olson C.A. Weinman O. Trester- Zedlitz M.L. Darst S.A. Mol. Cell. 2002; 9: 527-539Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). The subsequent structure of σ70 (regions 2-4) in complex with core RNA polymerase showed that these domains are modular, connected by flexible linkers, which interact with other parts of polymerase and DNA (16Murakami K.S. Masuda S. Darst S.A. Science. 2002; 296: 1280-1284Crossref PubMed Scopus (441) Google Scholar, 42Murakami K.S. Masuda S. Campbell E.A. Muzzin O. Darst S.A. Science. 2002; 296: 1285-1290Crossref PubMed Scopus (531) Google Scholar). Sequence analysis and biochemical experiments suggest that σ54 may also be comprised of such domains (43Sasse-Dwight S. Gralla J.D. Cell. 1990; 62: 945-954Abstract Full Text PDF PubMed Scopus (128) Google Scholar, 44Missailidis S. Cannon W.V. Drake A. Wang X.Y. Buck M. Mol. Microbiol. 1997; 24: 653-664Crossref PubMed Scopus (17) Google Scholar). Here we present the first structure of a σ54 domain, the C-terminal segment of the protein, which is critical for DNA binding and contains the signature “RpoN box” sequence (Fig. 1). Residues Gln-340 to Leu-388 of A. aeolicus σ54 form a compact, three-helix domain that includes a helix-turn-helix motif. A search of the Protein Data Bank using the DALI server found that this region of σ54 is structurally similar to many DNA-binding domains (supplemental Table S2). Of the ten structures with the highest Z scores, seven are known to interact with DNA. For two of these domains, the C-terminal part of the PAX6 homeodomain (PDB accession number 6PAX; Ref. 40Xu H.E. Rould M.A. Xu W. Epstein J.A. Maas R.L. Pabo C.O. Genes Dev. 1999; 13: 1263-1275Crossref PubMed Scopus (234) Google Scholar) and the N-terminal domain of the methicillin repressor protein (PDB accession number 1SAX; Ref. 45Garcia-Castellanos R. Marrero A. Mallorqui-Fernandez G. Potempa J. Coll M. Gomis-Ruth F.X. J. Biol. Chem. 2003; 278: 39897-39905Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), the structures of the protein-DNA complexes are known (40Xu H.E. Rould M.A. Xu W. Epstein J.A. Maas R.L. Pabo C.O. Genes Dev. 1999; 13: 1263-1275Crossref PubMed Scopus (234) Google Scholar, 46Garcia-Castellanos R. Mallorqui-Fernandez G. Marrero A. Potempa J. Coll M. Gomis-Ruth F.X. J. Biol. Chem. 2004; 279: 17888-17896Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). These structures show that the proteins interact primarily with the major groove of DNA via the second helix of the helix-turn-helix motif, termed the “recognition helix.” The equivalent helix in the σ54C domain is created by residues of the highly conserved RpoN box motif (helix 3; Figs. 2B and 3A). Thus, residues in the RpoN box are structurally poised to interact with the major groove of the σ54 promoter element. Consistent with interpreting structure of the σ54C domain as a DNA-binding domain, previous footprinting and gel shift assays have shown that the region near the RpoN box is critical for σ54 promoter recognition and DNA binding (19Cannon W.V. Chaney M.K. Wang X. Buck M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5006-5011Crossref PubMed Scopus (34) Google Scholar, 47Cannon W. Missailidis S. Smith C. Cottier A. Austin S. Moore M. Buck M. J. Mol. Biol. 1995; 248: 781-803Crossref PubMed Scopus (62) Google Scholar, 48Wang L. Gralla J.D. J. Biol. Chem. 2001; 276: 8979-8986Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Mutations in the RpoN box sequence are far more damaging to DNA binding than mutations in other parts of E. coli σ54 (21Taylor M. Butler R. Chambers S. Casimiro M. Badii F. Merrick M. Mol. Microbiol. 1996; 22: 1045-1054Crossref PubMed Scopus (44) Google Scholar, 48Wang L. Gralla J.D. J. Biol. Chem. 2001; 276: 8979-8986Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). All five mutations made in the RpoN box sequence had reduced binding to both duplex and fork junction promoter DNA, with four mutant proteins (K. pneumoniae R455A, R456A, Y461A, and R462A) showing ≤20% DNA binding of wild-type σ54 protein. In addition, these four mutant proteins had dramatically reduced transcription activity in vitro with ≤30% the activity of the wild-type protein. Thus, these residues on the RpoN box are important for DNA binding and σ54-dependent transcription. The corresponding residues in A. aeolicus σ54 are shown as sticks in Fig. 3A. Four of these residues (Arg-378, Arg-379, Lys-383, and Arg-385) lie on the hydrophilic face of the recognition helix, suggesting that this face of the helix interacts directly with DNA, as discussed in more detail later in this section. Additional support for the role of this domain in DNA binding comes from the work of Cannon et al. (47Cannon W. Missailidis S. Smith C. Cottier A. Austin S. Moore M. Buck M. J. Mol. Biol. 1995; 248: 781-803Crossref PubMed Scopus (62) Google Scholar). They found that removing the last 53 residues of K. pneumoniae σ54 (Lys-425 to Val-477; A. aeolicus Lys-346 to Ile-398) eliminated DNase I and 1,10-phenanthroline-copper footprints of σ54 and reduced σ54 holoenzyme binding to 5-bromouracil substituted DNA by 85%. This truncation cuts the protein in the middle of helix 1 of the σ54 C domain, removing loop 2 and the helix-turn-helix motif. Overall, the C-terminal domain of σ54 is more structurally similar to domain σ3 than to domain σ4 of σ70. σ3 and the σ54C are about the same size (55 and 59 residues, respectively), and all three helices are about the same length (Fig. 3B, right). On the other hand, σ4 has an extra helix at the N-terminal end, and the recognition helix is twice as long as the corresponding helix in the σ54C domain (Fig. 3B, left). In terms of surface charge distribution, however, the σ54C is more similar to σ4 than to σ3 (Fig. 3C). Both σ4 and σ54C have a positively charged surface created by side chains on the loop and the recognition helix of the helix-turn-helix motif (Fig. 3C, left and middle). For σ4, this surface interacts specifically with the major groove of the -35 promoter element (13Jain D. Nickels B.E. Sun L. Hochschild A. Darst S.A. Mol. Cell. 2004; 13: 45-53Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 15Campbell E.A. Muzzin O. Chlenov M. Sun J.L. Olson C.A. Weinman O. Trester- Zedlitz M.L. Darst S.A. Mol. Cell. 2002; 9: 527-539Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). For σ54C, this surface is created by the residues of the conserved RpoN box, which probably interact with the -24 promoter element. Previous biochemical studies suggest that the C-terminal domain of σ54 is more functionally analogous to the σ4 domain than to the σ3 domain of σ70-type factors. σ3 of σ70 proteins interacts with the extended -10 promoter element near the site of melting (42Murakami K.S. Masuda S. Campbell E.A. Muzzin O. Darst S.A. Science. 2002; 296: 1285-1290Crossref PubMed Scopus (531) Google Scholar, 49Voskuil M.I. Chambliss G.H. J. Mol. Biol. 2002; 322: 521-532Crossref PubMed Scopus (25) Google Scholar), whereas the major role of σ4 of σ70 proteins is to bind to the -35 promoter element (13Jain D. Nickels B.E. Sun L. Hochschild A. Darst S.A. Mol. Cell. 2004; 13: 45-53Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 15Campbell E.A. Muzzin O. Chlenov M. Sun J.L. Olson C.A. Weinman O. Trester- Zedlitz M.L. Darst S.A. Mol. Cell. 2002; 9: 527-539Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). By tethering a DNA cleavage reagent, FeBABE (p-bromoacetamidobenzyl-EDTA-Fe), to charged residues in the RpoN box of K. pneumoniae σ54, Burrows et al. (50Burrows P.C. Severinov K. Ishihama A. Buck M. Wigneshweraraj S.R. J. Biol. Chem. 2003; 278: 29728-29743Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) showed that residues in the RpoN box are near the -24 promoter element, not the -12 promoter element where melting occurs. For example, with the FeBABE tethered to residue E463C (A. aeolicus Glu-386), strong cleavage was seen between the -30 and -25 nucleotides of the template DNA strand. The N-terminal end of σ54C domain, residues Ser-325 to Thr-339, is not well ordered and probably dynamic on the microsecond-to-second time scale. These residues could be a part of a linker region that connects two independently folded domains of σ54, as seen with σ70-type factors (15Campbell E.A. Muzzin O. Chlenov M. Sun J.L. Olson C.A. Weinman O. Trester- Zedlitz M.L. Darst S.A. Mol. Cell. 2002; 9: 527-539Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 16Murakami K.S. Masuda S. Darst S.A. Science. 2002; 296: 1280-1284Crossref PubMed Scopus (441) Google Scholar). Previous limited proteolysis experiments on the full-length σ54 did not identify this segment as proteolytically sensitive (51Cannon W. Claverie-Martin F. Austin S. Buck M. Mol. Microbiol. 1994; 11: 227-236Crossref PubMed Scopus (40) Google Scholar). Thus, additional proteolysis studies and NMR analyses on larger fragments of the A. aeolicus σ54 are required to determine whether this region (Ser-325 to Thr-339) is still poorly structured in the presence of the other domains and RNAP. The C-terminal domain of σ54 presented in this report provides an interesting structural connection between σ54 and σ70. This structural similarity of σ54C to domains of σ70-type sigma factors is surprising (Fig. 3B) because these regions have poor sequence homology. Residues Thr-323 to Gly-389 of σ54C have 14% identity, 25% similarity with residues 186-326 of region 4 of σA from T. aquaticus and 15% identity, 26% similarity with residues 279-326 of region 3 of σ28 from A. aeolicus. A low resolution structure of the full-length σ54 protein suggests that other domains of σ54 may also be more similar to domains of σ70 than previously predicted by only sequence homology (22Svergun D.I. Malfois M. Koch M.H. Wigneshweraraj S.R. Buck M. J. Biol. Chem. 2000; 275: 4210-4214Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). High resolution structures of these other domains are needed to confirm this hypothesis. We thank Prof. Sydney Kustu for advice and Steven M. Damo for computer assistance. Download .pdf (1.77 MB) Help with pdf files" @default.
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• W2063452009 title "The C-terminal RpoN Domain of σ54 Forms an Unpredicted Helix-Turn-Helix Motif Similar to Domains of σ70" @default.
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