Abstract
λN-mediated processive antitermination constitutes a paradigmatic transcription regulatory event, during which phage protein λN, host factors NusA, NusB, NusE and NusG, and an RNA nut site render elongating RNA polymerase termination-resistant. The structural basis of the process has so far remained elusive. Here we describe a crystal structure of a λN–NusA–NusB–NusE–nut site complex and an electron cryo-microscopic structure of a complete transcription antitermination complex, comprising RNA polymerase, DNA, nut site RNA, all Nus factors and λN, validated by crosslinking/mass spectrometry. Due to intrinsic disorder, λN can act as a multiprotein/RNA interaction hub, which, together with nut site RNA, arranges NusA, NusB and NusE into a triangular complex. This complex docks via the NusA N-terminal domain and the λN C-terminus next to the RNA exit channel on RNA polymerase. Based on the structures, comparative crosslinking analyses and structure-guided mutagenesis, we hypothesize that λN mounts a multipronged strategy to reprogram the transcriptional machinery, which may include (1) the λN C terminus clamping the RNA exit channel, thus stabilizing the DNA:RNA hybrid; (2) repositioning of NusA and RNAP elements, thus redirecting nascent RNA and sequestering the upstream branch of a terminator hairpin; and (3) hindering RNA engagement of termination factor ρ and/or obstructing ρ translocation on the transcript.
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References
Zhang, J. & Landick, R. A two-way street: regulatory interplay between RNA polymerase and nascent RNA structure. Trends Biochem. Sci. 41, 293–310 (2016).
Mooney, R. A. et al. Regulator trafficking on bacterial transcription units in vivo. Mol. Cell 33, 97–108 (2009).
Yang, X. & Lewis, P. J. The interaction between RNA polymerase and the elongation factor NusA. RNA Biol. 7, 272–275 (2010).
Tomar, S. K. & Artsimovitch, I. NusG–Spt5 proteins—universal tools for transcription modification and communication. Chem. Rev. 113, 8604–8619 (2013).
Nudler, E. & Gottesman, M. E. Transcription termination and anti-termination in E. coli. Genes Cells 7, 755–768 (2002).
Ciampi, M. S. Rho-dependent terminators and transcription termination. Microbiology 152, 2515–2528 (2006).
Torres, M., Condon, C., Balada, J. M., Squires, C. & Squires, C. L. Ribosomal protein S4 is a transcription factor with properties remarkably similar to nusA, a protein involved in both non-ribosomal and ribosomal RNA antitermination. EMBO J. 20, 3811–3820 (2001).
Worbs, M., Bourenkov, G. P., Bartunik, H. D., Huber, R. & Wahl, M. C. An extended RNA binding surface through arrayed S1 and KH domains in transcription factor NusA. Mol. Cell 7, 1177–1189 (2001).
Luo, X. et al. Structural and functional analysis of the E. coli NusB–S10 transcription antitermination complex. Mol. Cell 32, 791–802 (2008).
Stagno, J. R. et al. Structural basis for RNA recognition by NusB and NusE in the initiation of transcription antitermination. Nucleic Acids Res. 39, 7803–7815 (2011).
Zhou, Y., Mah, T. F., Greenblatt, J. & Friedman, D. I. Evidence that the KH RNA-binding domains influence the action of the E. coli NusA protein. J. Mol. Biol. 318, 1175–1188 (2002).
Mogridge, J. et al. Independent ligand-induced folding of the RNA-binding domain and two functionally distinct antitermination regions in the phage lambda N protein. Mol. Cell 1, 265–275 (1998).
Legault, P., Li, J., Mogridge, J., Kay, L. E. & Greenblatt, J. NMR structure of the bacteriophage lambda N peptide/boxB RNA complex: recognition of a GNRA fold by an arginine-rich motif. Cell 93, 289–299 (1998).
Scharpf, M. et al. Antitermination in bacteriophage lambda. The structure of the N36 peptide–boxB RNA complex. Eur. J. Biochem. 267, 2397–2408 (2000).
Mogridge, J., Mah, T. F. & Greenblatt, J. Involvement of boxA nucleotides in the formation of a stable ribonucleoprotein complex containing the bacteriophage lambda N protein. J. Biol. Chem. 273, 4143–4148 (1998).
Mah, T. F., Li, J., Davidson, A. R. & Greenblatt, J. Functional importance of regions in Escherichia coli elongation factor NusA that interact with RNA polymerase, the bacteriophage lambda N protein and RNA. Mol. Microbiol. 34, 523–537 (1999).
Bonin, I. et al. Structural basis for the interaction of Escherichia coli NusA with protein N of phage lambda. Proc. Natl. Acad. Sci. USA. 101, 13762–13767 (2004).
Mishra, S., Mohan, S., Godavarthi, S. & Sen, R. The interaction surface of a bacterial transcription elongation factor required for complex formation with an antiterminator during transcription antitermination. J. Biol. Chem. 288, 28089–28103 (2013).
Mah, T. F., Kuznedelov, K., Mushegian, A., Severinov, K. & Greenblatt, J. The alpha subunit of E. coli RNA polymerase activates RNA binding by NusA. Genes Dev. 14, 2664–2675 (2000).
Mogridge, J., Mah, T. F. & Greenblatt, J. A protein–RNA interaction network facilitates the template-independent cooperative assembly on RNA polymerase of a stable antitermination complex containing the lambda N protein. Genes Dev. 9, 2831–2845 (1995).
Zhou, Y. et al. Interactions of an Arg-rich region of transcription elongation protein NusA with NUT RNA: implications for the order of assembly of the lambda N antitermination complex in vivo. J. Mol. Biol. 310, 33–49 (2001).
Friedman, D. I. & Baron, L. S. Genetic characterization of a bacterial locus involved in the activity of the N function of phage lambda. Virology 58, 141–148 (1974).
Ward, D. F. & Gottesman, M. E. The nus mutations affect transcription termination in Escherichia coli. Nature 292, 212–215 (1981).
Craven, M. G. & Friedman, D. I. Analysis of the Escherichia coli nusA10(Cs) allele: relating nucleotide changes to phenotypes. J. Bacteriol. 173, 1485–1491 (1991).
Craven, M. G. et al. Escherichia coli–Salmonella typhimurium hybrid nusA genes: identification of a short motif required for action of the lambda N transcription antitermination protein. J. Bacteriol. 176, 1394–1404 (1994).
Friedman, D. I. et al. Interactions of bacteriophage and host macromolecules in the growth of bacteriophage lambda. Microbiol. Rev. 48, 299–325 (1984).
Ward, D. F., DeLong, A. & Gottesman, M. E. Escherichia coli nusB mutations that suppress nusA1 exhibit lambda N specificity. J. Mol. Biol. 168, 73–85 (1983).
Friedman, D. I., Schauer, A. T., Baumann, M. R., Baron, L. S. & Adhya, S. L. Evidence that ribosomal protein S10 participates in control of transcription termination. Proc. Natl Acad. Sci. USA 78, 1115–1118 (1981).
Sparkowski, J. & Das, A. Simultaneous gain and loss of functions caused by a single amino acid substitution in the beta subunit of Escherichia coli RNA polymerase: suppression of nusA and rho mutations and conditional lethality. Genetics 130, 411–428 (1992).
Friedman, D. I. & Olson, E. R. Evidence that a nucleotide sequence, ‘boxA,’ is involved in the action of the NusA protein. Cell 34, 143–149 (1983).
Friedman, D. I., Olson, E. R., Johnson, L. L., Alessi, D. & Craven, M. G. Transcription-dependent competition for a host factor: the function and optimal sequence of the phage lambda boxA transcription antitermination signal. Genes Dev. 4, 2210–2222 (1990).
Liu, B. & Steitz, T. A. Structural insights into NusG regulating transcription elongation. Nucleic Acids Res. 45, 968–974 (2017).
Martinez-Rucobo, F. W., Sainsbury, S., Cheung, A. C. M. & Cramer, P. Architecture of the RNA polymerase-Spt4/5 complex and basis of universal transcription processivity. EMBO J. 30, 1302–1310 (2011).
Burmann, B. M. et al. A NusE:NusG complex links transcription and translation. Science 328, 501–504 (2010).
Sullivan, S. L., Ward, D. F. & Gottesman, M. E. Effect of Escherichia coli nusG function on lambda N-mediated transcription antitermination. J. Bacteriol. 174, 1339–1344 (1992).
Kuznedelov, K., Korzheva, N., Mustaev, A. & Severinov, K. Structure-based analysis of RNA polymerase function: the largest subunit's rudder contributes critically to elongation complex stability and is not involved in the maintenance of RNA–DNA hybrid length. EMBO J. 21, 1369–1378 (2002).
Gusarov, I. & Nudler, E. Control of intrinsic transcription termination by N and NusA: the basic mechanisms. Cell 107, 437–449 (2001).
Parks, A. R. et al. Bacteriophage lambda N protein inhibits transcription slippage by Escherichia coli RNA polymerase. Nucleic Acids Res. 42, 5823–5829 (2014).
Mishra, S. & Sen, R. N protein from lambdoid phages transforms NusA into an antiterminator by modulating NusA–RNA polymerase flap domain interactions. Nucleic Acids Res. 43, 5744–5758 (2015).
Cheeran, A. et al. Escherichia coli RNA polymerase mutations located near the upstream edge of an RNA:DNA hybrid and the beginning of the RNA-exit channel are defective for transcription antitermination by the N protein from lambdoid phage H-19B. J. Mol. Biol. 352, 28–43 (2005).
Georgopoulos, C. P. Bacterial mutants in which gene N function of bacteriophage lambda is blocked have an altered RNA polymerase. Proc. Natl Acad. Sci. USA 68, 2977–2981 (1971).
Jin, D. J. et al. Effects of rifampicin resistant rpoB mutations on antitermination and interaction with nusA in Escherichia coli. J. Mol. Biol. 204, 247–261 (1988).
Cheeran, A., Kolli, N. R. & Sen, R. The site of action of the antiterminator protein N from the lambdoid phage H-19B. J. Biol. Chem. 282, 30997–31007 (2007).
Murakami, K. S. X-ray crystal structure of Escherichia coli RNA polymerase sigma70 holoenzyme. J. Biol. Chem. 288, 9126–9134 (2013).
Toulokhonov, I., Artsimovitch, I. & Landick, R. Allosteric control of RNA polymerase by a site that contacts nascent RNA hairpins. Science 292, 730–733 (2001).
Ha, K. S., Toulokhonov, I., Vassylyev, D. G. & Landick, R. The NusA N-terminal domain is necessary and sufficient for enhancement of transcriptional pausing via interaction with the RNA exit channel of RNA polymerase. J. Mol. Biol. 401, 708–725 (2010).
Rees, W. A., Weitzel, S. E., Yager, T. D., Das, A. & von Hippel, P. H. Bacteriophage lambda N protein alone can induce transcription antitermination in vitro. Proc. Natl Acad. Sci. USA 93, 342–346 (1996).
Drogemuller, J. et al. Determination of RNA polymerase binding surfaces of transcription factors by NMR spectroscopy. Sci. Rep. 5, 16428 (2015).
Kolb, K. E., Hein, P. P. & Landick, R. Antisense oligonucleotide-stimulated transcriptional pausing reveals RNA exit channel specificity of RNA polymerase and mechanistic contributions of NusA and rfaH. J. Biol. Chem. 289, 1151–1163 (2014).
Beuth, B., Pennell, S., Arnvig, K. B., Martin, S. R. & Taylor, I. A. Structure of a Mycobacterium tuberculosis NusA–RNA complex. EMBO J. 24, 3576–3587 (2005).
Artsimovitch, I., Svetlov, V., Murakami, K. S. & Landick, R. Co-overexpression of Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions. J. Biol. Chem. 278, 12344–12355 (2003).
Studier, F. W. Protein production by auto-induction in high-density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).
Kyzer, S., Ha, K. S., Landick, R. & Palangat, M. Direct versus limited-step reconstitution reveals key features of an RNA hairpin-stabilized paused transcription complex. J. Biol. Chem. 282, 19020–19028 (2007).
Belogurov, G. A. et al. Structural basis for converting a general transcription factor into an operon-specific virulence regulator. Mol. Cell 26, 117–129 (2007).
Easton, L. E., Shibata, Y. & Lukavsky, P. J. Rapid, nondenaturing RNA purification using weak anion-exchange fast performance liquid chromatography. RNA 16, 647–653 (2010).
McKenna, S. A. et al. Purification and characterization of transcribed RNAs using gel filtration chromatography. Nat. Protoc. 2, 3270–3277 (2007).
Kao, C., Zheng, M. & Rudisser, S. A simple and efficient method to reduce nontemplated nucleotide addition at the 3 terminus of RNAs transcribed by T7 RNA polymerase. RNA 5, 1268–1272 (1999).
Sherlin, L. D. et al. Chemical and enzymatic synthesis of tRNAs for high-throughput crystallization. RNA 7, 1671–1678 (2001).
Rao, R. N. Construction and properties of plasmid pKC30, a pBR322 derivative containing the pL-N region of phage lambda. Gene 31, 247–250 (1984).
Artsimovitch, I. & Henkin, T. M. In vitro approaches to analysis of transcription termination. Methods 47, 37–43 (2009).
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).
Shin, D. H. et al. Crystal structure of NusA from Thermotoga maritima and functional implication of the N-terminal domain. Biochemistry 42, 13429–13437 (2003).
McCoy, A. J. Solving structures of protein complexes by molecular replacement with phaser. Acta Crystallogr. D 63, 32–41 (2007).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of coot. Acta Crystallogr. D 66, 486–501 (2010).
Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).
Schroder, G. F., Levitt, M. & Brunger, A. T. Deformable elastic network refinement for low-resolution macromolecular crystallography. Acta Crystallogr. D 70, 2241–2255 (2014).
Vassylyev, D. G., Vassylyeva, M. N., Perederina, A., Tahirov, T. H. & Artsimovitch, I. Structural basis for transcription elongation by bacterial RNA polymerase. Nature 448, 157–162 (2007).
Vassylyev, D. G. et al. Structural basis for substrate loading in bacterial RNA polymerase. Nature 448, 163–168 (2007).
Westover, K. D., Bushnell, D. A. & Kornberg, R. D. Structural basis of transcription: separation of RNA from DNA by RNA polymerase II. Science 303, 1014–1016 (2004).
Westover, K. D., Bushnell, D. A. & Kornberg, R. D. Structural basis of transcription: nucleotide selection by rotation in the RNA polymerase II active center. Cell 119, 481–489 (2004).
Kettenberger, H., Armache, K. J. & Cramer, P. Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. Mol. Cell 16, 955–965 (2004).
Suloway, C. et al. Automated molecular microscopy: the new leginon system. J. Struct. Biol. 151, 41–60 (2005).
Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).
Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).
Yang, Z., Fang, J., Chittuluru, J., Asturias, F. J. & Penczek, P. A. Iterative stable alignment and clustering of 2D transmission electron microscope images. Structure 20, 237–247 (2012).
Penczek, P. A. & Asturias, F. J. Ab initio cryo-EM structure determination as a validation problem. Proc. IEEE Int. Conf. Image Processing (ICIP) 2014, 2090–2094 (2014).
Cheng, Y., Grigorieff, N., Penczek, P. A. & Walz, T. A primer to single-particle cryo-electron microscopy. Cell 161, 438–449 (2015).
Hohn, M. et al. SPARX, a new environment for cryo-EM image processing. J. Struct. Biol. 157, 47–55 (2007).
Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).
Scheres, S. H. Semi-automated selection of cryo-EM particles in RELION-1.3. J. Struct. Biol. 189, 114–122 (2015).
Bharat, T. A., Russo, C. J., Lowe, J., Passmore, L. A. & Scheres, S. H. Advances in single-particle electron cryomicroscopy structure determination applied to sub-tomogram averaging. Structure 23, 1743–1753 (2015).
Frank, J., Shimkin, B. & Dowse, H. Spider—a modular software system for electron image-processing. Ultramicroscopy 6, 343–357 (1981).
Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).
Liu, B., Zuo, Y. & Steitz, T. A. Structural basis for transcription reactivation by RapA. Proc. Natl Acad. Sci. USA 112, 2006–2010 (2015).
Bernecky, C., Herzog, F., Baumeister, W., Plitzko, J. M. & Cramer, P. Structure of transcribing mammalian RNA polymerase II. Nature 529, 551–554 (2016).
Mooney, R. A., Schweimer, K., Rosch, P., Gottesman, M. & Landick, R. Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators. J. Mol. Biol. 391, 341–358 (2009).
Schmidt, C. & Urlaub, H. iTRAQ-labeling of in-gel digested proteins for relative quantification. Methods Mol. Biol. 564, 207–226 (2009).
Leitner, A., Walzthoeni, T. & Aebersold, R. Lysine-specific chemical cross-linking of protein complexes and identification of cross-linking sites using LC-MS/MS and the xQuest/xProphet software pipeline. Nat. Protoc. 9, 120–137 (2014).
Yang, B. et al. Identification of cross-linked peptides from complex samples. Nat. Methods 9, 904–906 (2012).
Acknowledgements
The authors thank C. Alings (Freie Universität Berlin) for help with crystallization, I. Artsimovitch (Ohio State University) for plasmids pVS-10 (T7P-α-β-β′His6-ω) and pIA1127 (T7P-His6-TEV-σ70), used for RNAP production, and M. Gottesman (New York University) for plasmid pKC30, used in transcription assays. The authors acknowledge access to beamlines of the BESSY II storage ring (Berlin, Germany) via the Joint Berlin MX-Laboratory sponsored by the Helmholtz Zentrum Berlin für Materialien und Energie, the Freie Universität Berlin, the Humboldt-Universität zu Berlin, the Max-Delbrück-Centrum and the Leibniz-Institut für Molekulare Pharmakologie and to beamline 14–1 at the Petra III storage ring (EMBL, Hamburg, Germany). The authors acknowledge access to the resources of the North-German Supercomputing Alliance (HLRN). This work was supported by the Deutsche Forschungsgemeinschaft (SFB 740 to T.M., C.M.S. and M.C.W. and grant WA 1126/5-1 to M.C.W.). K.F.S. was supported by a Dahlem International Network PostDoc grant from Freie Universität Berlin. E.B. holds a Freigeist Fellowship from the Volkswagen Foundation and acknowledges continuing support from the Caesar Foundation.
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N.S., F.K., E.A., K.F.S., O.D., Y.-H.H., C.-T.L., B.L., E.B., J.B., T.M., J.L. and G.W. performed the experiments. All authors contributed to analysis of the data and interpretation of the results. H.U., C.M.T.S. and M.C.W. supervised the studies. N.S. and M.C.W. wrote the manuscript.
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Supplementary Discussion, Supplementary Figures 1 and 2, Supplementary Tables 1 and 2, Supplementary References. (PDF 1890 kb)
Supplementary Table 3
Chemical CX-MS of a λN-based transcription antitermination complex (TAC). (XLSX 39 kb)
Supplementary Table 4
Chemical CX-MS of a transcription elongation complex (TEC) lacking λN. (XLSX 29 kb)
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Said, N., Krupp, F., Anedchenko, E. et al. Structural basis for λN-dependent processive transcription antitermination. Nat Microbiol 2, 17062 (2017). https://doi.org/10.1038/nmicrobiol.2017.62
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DOI: https://doi.org/10.1038/nmicrobiol.2017.62
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