Structural basis for λN-dependent processive transcription antitermination

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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Figure 1: Structure of a λN1–84–NusAΔAR2–NusB–NusE–nut RNP.
Figure 2: Interaction studies.
Figure 3: Mapping of subunit variants.
Figure 4: Structure of a λN-based transcription antitermination complex.
Figure 5: Mechanism of λN-mediated processive antitermination.
Figure 6: In vitro functional analysis of antitermination activity.

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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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Correspondence to Markus C. Wahl.

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Supplementary information

Supplementary Information

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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