Solution Structure of YaeO, a Rho-specific Inhibitor of Transcription Termination

Rho-dependent transcription termination is an essential process for the regulation of bacterial gene expression. Thus far, only two Rho-specific inhibitors of bacterial transcription termination have been described, the psu protein from the satellite bacteriophage P4 and YaeO from Escherichia coli. Here, we report the solution structure of YaeO, the first of a Rho-specific inhibitor of transcription termination. YaeO is an acidic protein composed of an N-terminal helix and a seven-stranded β sandwich. NMR chemical shift perturbation experiments revealed that YaeO binds proximal to the primary nucleic acid binding site of Rho. Based on the NMR titrations, a docked model of the YaeO-Rho complex was calculated. These results suggest that YaeO binds outside the Rho hexamer, acting as a competitive inhibitor of RNA binding. In vitro gel shift assays confirmed the inhibition of nucleic acid binding to Rho. Site-directed mutagenesis showed that the negative character of YaeO is essential for its function in vivo. Rho-dependent transcription termination is an essential process for the regulation of bacterial gene expression. Thus far, only two Rho-specific inhibitors of bacterial transcription termination have been described, the psu protein from the satellite bacteriophage P4 and YaeO from Escherichia coli. Here, we report the solution structure of YaeO, the first of a Rho-specific inhibitor of transcription termination. YaeO is an acidic protein composed of an N-terminal helix and a seven-stranded β sandwich. NMR chemical shift perturbation experiments revealed that YaeO binds proximal to the primary nucleic acid binding site of Rho. Based on the NMR titrations, a docked model of the YaeO-Rho complex was calculated. These results suggest that YaeO binds outside the Rho hexamer, acting as a competitive inhibitor of RNA binding. In vitro gel shift assays confirmed the inhibition of nucleic acid binding to Rho. Site-directed mutagenesis showed that the negative character of YaeO is essential for its function in vivo. Transcription termination is the process by which a nascent RNA is released from its complex with RNA polymerase and DNA template. In bacteria, two main mechanisms of transcription termination have been described. These mechanisms, commonly referred to as Rho-independent and Rho-dependent termination, are essential for the regulation of bacterial gene expression (1Richardson J.P. Greenblatt J. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. 1996; (Neidhardt, F. C., ed) pp. American Society for Microbiology Press, Washington, D.C.: 822-848Google Scholar). Rho-independent termination occurs at a GC-rich self-complementarity region that forms a stem-loop structure believed to cause the RNA polymerase to pause, allowing the release of the RNA (2Brendel V. Hamm G.H. Trifonov E.N. J. Biomol. Struct. Dyn. 1986; 3: 705-723Crossref PubMed Scopus (80) Google Scholar, 3Rosenberg M. Court D. Annu. Rev. Genet. 1979; 13: 319-353Crossref PubMed Scopus (1702) Google Scholar). Rho-dependent termination, on the other hand, requires the presence of a hexameric helicase, Rho (4Brown S. Brickman E.R. Beckwith J. J. Bacteriol. 1981; 146: 422-425Crossref PubMed Google Scholar, 5Opperman T. Richardson J.P. J. Bacteriol. 1994; 176: 5033-5043Crossref PubMed Google Scholar). Rho is an essential transcription factor that binds nucleic acids at specific termination sites (rut) and translocates along the RNA until it reaches the transcription complex (6Geiselmann J. Wang Y. Seifried S.E. von Hippel P.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7754-7758Crossref PubMed Scopus (101) Google Scholar, 7Platt T. Mol. Microbiol. 1994; 11: 983-990Crossref PubMed Scopus (83) Google Scholar, 8Richardson J.P. J. Biol. Chem. 1996; 271: 1251-1254Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). There, it facilitates termination by unwinding RNA/DNA heteroduplexes upon hydrolysis of ATP (9Brennan C.A. Dombroski A.J. Platt T. Cell. 1987; 48: 945-952Abstract Full Text PDF PubMed Scopus (186) Google Scholar). Currently, only two Rho-specific inhibitors of transcription termination have been reported. The first to be described is a 21.3-kDa protein encoded by gene psu of the satellite bacteriophage P4 (10Linderoth N.A. Calendar R.L. J. Bacteriol. 1991; 173: 6722-6731Crossref PubMed Google Scholar). Psu interferes with transcription in phage, plasmid, and bacterial operons, and its activity does not depend on sequences in the transcript. In vitro, protein Psu causes efficient readthrough of Rho-dependent terminators λ tR1 and TIS2 in a manner that seems to be insensitive to NusG (11Linderoth N.A. Tang G. Calendar R. Virology. 1997; 227: 131-141Crossref PubMed Scopus (13) Google Scholar). Whether Psu inhibits an enzymatic activity of Rho or the interaction of Rho with RNA, ATP, NusG, or RNA polymerase is unknown. NusG is a transcriptional elongation factor that interacts with both Rho and RNA polymerase (12Pasman Z. von Hippel P.H. Biochemistry. 2000; 39: 5573-5585Crossref PubMed Scopus (71) Google Scholar, 13Li J. Mason S.W. Greenblatt J. Genes Dev. 1993; 7: 161-172Crossref PubMed Scopus (110) Google Scholar). The second inhibitor is the product of gene yaeO from Escherichia coli, which has been shown to reduce termination in the Rho-dependent bacteriophage terminator tL1 and upstream the autogenously regulated gene rho (14Pichoff S. Alibaud L. Guédant A. Castanié M.P. Bouché J.P. Mol. Microbiol. 1998; 29: 859-869Crossref PubMed Scopus (28) Google Scholar). Overexpression of YaeO can cause the pleiotropic suppression of conditional lethal mutations in division and heat shock genes such as ftsQ, ftsA, grpE, groEL, and groES (14Pichoff S. Alibaud L. Guédant A. Castanié M.P. Bouché J.P. Mol. Microbiol. 1998; 29: 859-869Crossref PubMed Scopus (28) Google Scholar). YaeO is a 9-kDa acidic protein that binds tightly to Rho, but the exact nature of this interaction was unknown (14Pichoff S. Alibaud L. Guédant A. Castanié M.P. Bouché J.P. Mol. Microbiol. 1998; 29: 859-869Crossref PubMed Scopus (28) Google Scholar). Here, the solution structure of YaeO is reported, the first of a Rho-specific inhibitor of transcription termination. Additionally, the binding surface of the Rho-YaeO complex was mapped for both proteins and a docked model calculated. Finally, the effect of some YaeO mutants on Rho-dependent transcription termination was tested in vivo and a mechanism for YaeO-mediated regulation proposed. Sample Preparation—The gene yaeO from E. coli K12 was subcloned into pET15b (Novagen, Inc., Madison, WI) and expressed in E. coli BL21 as an oligo-histidine (His tag) fusion protein of 106 residues. Cells were grown at 37 °C to an A600 of 0.8 and induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside. Afterward, the temperature was reduced to 30 °C and the cells were allowed to express the protein for 3 h before harvesting. The media used were either Luria Bertani or M9 minimal medium containing [15N] ammonium chloride and/or [13C]glucose (Cambridge Isotopes Laboratory, Andover, MA). YaeO was purified by affinity chromatography on Ni2+-loaded chelating Sepharose (Amersham Biosciences). NMR samples were ∼2 mm protein in 50 mm phosphate buffer 1 mm NaN3, 2 mm dithiothreitol, pH 7.0. Full-length Rho and the amino-terminal domain of Rho, residues 1–130 (Rho130), were cloned, expressed, and purified in a similar fashion. For simplicity in the text, residues in YaeO and Rho are numbered according to the wild-type protein sequences. NMR Spectroscopy—NMR experiments were recorded at 303 K on a Bruker Avance 600 MHz spectrometer. Backbone and side-chain assignments of YaeO were determined using HNCACB, CBCA(CO)NH, 15N-edited TOCSY, and 13C-edited TOCSY. Nuclear Overhauser effect (NOE) 2The abbreviations used are: NOE, nuclear Overhauser effect; HSQC, heteronuclear single quantum correlation; r.m.s., root mean square. data for the structure determination were obtained from homonuclear NOESY, 15N-edited or 13C-edited three-dimensional NOESY experiments. Evaluation of spectra and manual assignments was completed with XEASY (15Bartels C. Xia T. Billeter M. Guntert P. Wuöthrich K. J. Biomol. NMR. 1995; 6: 1-10Crossref PubMed Scopus (1604) Google Scholar). IPAP-HSQC experiments for measuring 15N-1H dipolar couplings were recorded on an isotropic medium and a sample containing 18 mg/ml Pf1 phage (16Ottiger M. Delaglio F. Bax A. J. Magn. Reson. 1998; 131: 373-378Crossref PubMed Scopus (842) Google Scholar, 17Hansen M.R. Mueller L. Pardi A. Nat. Struct. Biol. 1998; 5: 1065-1074Crossref PubMed Scopus (690) Google Scholar). 15N{1H}-heteronuclear NOE data were measured by taking the ratio of peak intensities from experiments performed with and without 1H presaturation. Hydrogen bond constraints were introduced to secondary structure regions as determined by chemical shift analysis and characteristic NOE patterns. ϕ and Ψ dihedral restraints were obtained using the TALOS program (18Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar). All NMR spectra were processed using either XWINNMR version 2.5 or 3.1 (Bruker Biospin) or GIFA (19Malliavin T.E. Pons J.L. Delsuc M.A. Bioinformatics. 1998; 14: 624-631Crossref PubMed Scopus (21) Google Scholar). Evaluation of spectra and manual assignments was completed with XEASY (15Bartels C. Xia T. Billeter M. Guntert P. Wuöthrich K. J. Biomol. NMR. 1995; 6: 1-10Crossref PubMed Scopus (1604) Google Scholar). Analysis and Structure Calculations—CNS 1.1 software (20Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54 (, Pt. 5,): 905-921Crossref PubMed Scopus (16967) Google Scholar) was used to generate an initial fold of YaeO with a basic set of manually assigned NOEs obtained from three-dimensional 15N-edited NOESY and two-dimensional homonuclear NOE spectra and dihedral angle and hydrogen bond constraints (21Wuöthrich K. NMR of Proteins and Nucleic Acids. 1986; (, Wiley & Sons, New York)Google Scholar). These calculations generated a fold that was used as a model template for automated assignments by ARIA 1.1 (22Nilges M. Macias M.J. O'Donoghue S.I. Oschkinat H. J. Mol. Biol. 1997; 269: 408-422Crossref PubMed Scopus (388) Google Scholar). The final structure of YaeO was calculated using the constraints in Table 1 and collected from the experiments described above. In the final round of calculations, CNS 1.1 was extended to incorporate residual dipolar coupling restraints (RDCs) for further refinement. The axial and rhombic components of the alignment tensor were defined from a histogram of measured RDCs (23Clore G.M. Gronenborn A.M. Bax A. J. Magn. Reson. 1998; 133: 216-221Crossref PubMed Scopus (334) Google Scholar) and optimized by a grid search method (24Clore G.M. Gronenborn A.M. Tjandra N. J. Magn. Reson. 1998; 131: 159-162Crossref PubMed Scopus (278) Google Scholar). Twenty structures were selected based on the lowest overall energy and least violations to represent final structures. PROCHECK-NMR was used to generate Ramachandran plots to check the stereochemical geometry of the protein (25Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar).TABLE 1Structural statistics for 20 selected conformers for YaeOConstraints used for structure calculationIntraresidue NOEs(n = 0)270Sequential range NOEs(n = 1)253Medium range NOEs(n = 2,3,4)68Long range NOEs(n > 4)159Dihedral angle constraints107Hydrogen bonds3315N-1H residual dipolar couplings61Total number of constraints951Final energies (kcal/mol)Etotal265.67 ± 4.57Ebond8.76 ± 0.55Eangle96.70 ± 2.65Eimproper19.52 ± 1.02EVdW80.94 ± 3.39Enoe30.95 ± 1.68Edihedral9.02 ± 0.61Esani19.75 ± 1.53r.m.s. deviation from idealized covalent geometryBonds (Å)0.0025 ± 0.00001Angles (°)0.5081 ± 0.0072Impropers (°)0.4390 ± 0.01134Average r.m.s. difference to mean structure (Å)Backbone atoms0.40 ± 0.12All heavy (non-hydrogen) atoms1.04 ± 0.10All atoms1.22 ± 0.08r.m.s. deviation from NMR restraintsDistance restraints (Å)0.0241 ± 0.0007Dihedral angle restraints (°)0.8315 ± 0.0281Average Ramachandran statistics (%)Residues in most favored regions87.1Residues in additional allowed regions10.8Residues in generously allowed regions2.1Residues in disallowed regions0.0Analysis of residual dipolar couplingsr.m.s. deviation (Hz)1.390 ± 0.035Q-factor0.138 ± 0.003 Open table in a new tab Ligand Titration—Chemical shift perturbation analyses were performed by recording a series of 15N-HSQC spectra at 298 K on uniformly 15N-labeled YaeO or Rho130 (∼2 mm) in the presence of different amounts of ligand. The protein sample and stock solutions of the ligands were all prepared for NMR as described in sample preparation. Docking—Docking of YaeO and Rho130 was done using HADDOCK (26Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2189) Google Scholar). Ambiguous interaction restraints were defined for residues at least 50% solvent-exposed and with chemical shift perturbations above the average. Mobile regions were determined based on heteronuclear NOE data. 200 structures were calculated during the first step of rigid body energy minimization followed by 50 structures of semirigid simulated annealing in torsion angle space. The best model was selected based on the convergence of the structures at the interface and intermolecular energy. Site-directed Mutagenesis—For site-directed mutagenesis, plasmid pSEB41 was used as a template. Acidic residues from YaeO thought to be involved in the interaction with Rho were mutated to lysine using the QuikChange site-directed mutagenesis kit (Stratagene). Mutations were confirmed by DNA sequencing. β-Galactosidase Assays—Strains JS219/pJPB314 (lacZp) and JS219/pJPB314/pSEB41 (lacZp-yaeO) were grown at 37 °C in Luria broth supplemented with 2% glucose. Cultures were maintained below A600 = 1 by dilution. Strain JS219/pJPB314 was also transformed with plasmid pSEB41 containing D14K, E40K, or E73K mutations. β-Galactosidase assays were performed as described by Miller (27Miller J.H. Experiments in Molecular Genetics. 1972; (Miller, J. H., ed) pp. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY: 352-355Google Scholar). Strains and plasmids were kindly provided by Dr. J-P. Bouché (CNRS, Toulouse Cédex, France). Electromobility Gel Shift Assays—Assays of nucleic acid binding to full-length Rho were carried out as described in Ref. 28Pani B. Banerjee S. Chalissery J. Abishek M. Loganathan R.M. Suganthan R.B. Sen R. J. Biol. Chem. 2006; 281: 26491-26500Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar. Chemically synthesized oligo(dC)34 was 32P-labeled with T4 polynucleotide kinase and mixed with the indicated proteins in 25 mm Tris-HCl, pH 8.0, 5 mm MgCl, and 50 mm KCl, 10% glycerol, and 0.2 mg/ml bovine serum albumin for 5 min before on a gel with in buffer mm mm 2 mm and Structure of was in E. coli as an N-terminal fusion protein and purified by affinity The was not for as its presence not the structure of the as by of spectra of the and The protein was uniformly with or with and for NMR Backbone assignments were obtained with NMR experiments. The overall structure of YaeO is defined by the NMR for residues and which are not These residues have NOEs and NOEs to which is of in solution secondary chemical shift we that YaeO is composed of helix and a seven-stranded The of secondary structure to the primary that helix is the best secondary structure in the and in residues at helix or the of and are The of residues are in the surface of YaeO, that 1 a of the lowest energy NMR structures. The region residues The two of the are by β and β residues and residues both to a which and The regions of YaeO have root mean of for and for all heavy The surface of YaeO that with and forms a to residues and Analysis of to be a of a of inhibitors of Rho-dependent transcription termination. proteins are in of and with a of a and of the with the program revealed similar structures with a The most of are the pleiotropic the protein the nuclear and the were and with r.m.s. from to for These results the of the fold of YaeO is similar to that of the RNA binding domain of The most difference the and YaeO is the presence of an additional in is to most of the are transcriptional or involved in with DNA performed on YaeO not interaction not YaeO and the mechanism of transcription termination inhibition by YaeO, NMR experiments were performed to the interaction of YaeO with Rho in For a version of Rho, from E. coli was to the primary RNA binding site of Rho and has been shown to be a model of Richardson J.P. Nat. Struct. Biol. 1998; 5: PubMed Scopus Google Scholar). performed an NMR by recording a series of spectra of 15N-labeled YaeO as a function of Rho130 as by chemical shift perturbations in the spectra of the and spectra allowed to the binding site of Rho on YaeO and The chemical in the and helix and and These regions to of the with acidic residues. These results suggest that the and of YaeO upon binding to Rho. the structure of Rho130 has been by NMR Richardson J.P. Nat. Struct. Biol. 1998; 5: PubMed Scopus Google we to the interaction with YaeO on Rho130 Rho130 was uniformly 15N-labeled and the were assigned using the chemical for the determined NMR kindly provided by Dr. G. Richardson J.P. J. Biomol. NMR. 1996; PubMed Google Scholar). Rho130 is composed of an and a shift perturbation analysis showed that YaeO binds to and helix of the were also for helix 3 and 1 and has shown that RNA binds to the N-terminal domain of Rho by with and E. J.M. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Richardson Richardson J.P. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar, E. J.M. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). These residues are of the region of Rho, a mechanism of transcription termination inhibition by the RNA binding site on Rho of the Rho-YaeO NMR data for the YaeO-Rho interaction were for both a model of the complex was using protein (26Dominguez C. Boelens R. Bonvin A.M. J. Am. Chem. Soc. 2003; 125: 1731-1737Crossref PubMed Scopus (2189) Google Scholar). Ambiguous interaction restraints were from the NMR data by residues with both the chemical and residues were allowed to during the The model with the lowest intermolecular energy the of in with was The interaction YaeO and Rho seems to be by and of of surface is with in vitro binding results that the YaeO-Rho interaction is and can be at (14Pichoff S. Alibaud L. Guédant A. Castanié M.P. Bouché J.P. Mol. Microbiol. 1998; 29: 859-869Crossref PubMed Scopus (28) Google Scholar). The model and The model is with the hexameric structure Rho as are the model is to the structure of Rho YaeO binding with the of RNA to Rho and is to binding. YaeO of Nucleic by this we carried out gel shift assays with oligo(dC)34 and Rho in the presence and of The binding of by hexameric Rho is competitive with the binding of Richardson J.P. J. Mol. Biol. PubMed Scopus Google Scholar). of 5 YaeO of complex at two different of Rho a of YaeO to Rho, DNA binding was of the Rho model of YaeO to Rho the of the two protein the of the YaeO D14K, and were The of mutations were tested in the gel shift All mutations the inhibition of nucleic acid binding to Rho The and of the proteins were by NMR The mutants were also tested in vivo using a by (14Pichoff S. Alibaud L. Guédant A. Castanié M.P. Bouché J.P. Mol. Microbiol. 1998; 29: 859-869Crossref PubMed Scopus (28) Google Scholar). In this the expression of is the of a Rho binding upstream of the of YaeO inhibits Rho, allowing the of of The mutants tested showed similar to the negative that the acidic residues are for YaeO inhibition of Rho data that YaeO binds to the transcription factor Rho and a for its inhibition of transcription termination. is that YaeO binds to the Rho in a Gel shift assays that YaeO the affinity of Rho for nucleic acids with an in vitro for YaeO of 5 have that the of RNA binding to hexameric Rho of and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (37) Google Scholar). is the RNA binds to the primary binding site of Rho. RNA the binding sites in the to The step of the of the to of the RNA the to Finally, is the Rho in and data suggest that the YaeO by the of the and/or Rho is to and essential for the of bacterial as a it is an for The of Rho from the of in from and T. S. M. Y. J. 1972; PubMed Scopus Google Scholar, S. N. H. S. H. J. 1972; PubMed Scopus Google Scholar). activity such as E. coli, and Salmonella and such as A. H. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The mechanism of YaeO is different from that of as the binds to the domain of Rho E. H. J.M. 13: Full Text Full Text PDF PubMed Scopus Google Scholar). The of the structure of YaeO and its binding site on Rho a new for the of Rho. YaeO the of a of that the initial in the of Rho with S. of for kindly the chemical of Rho130 and Bouché for the for the in vivo YaeO also and for and with

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