Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA.
Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3' terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3' terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3' cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3' termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3' terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3' vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provide insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1 3PU, UK nicole.robb@physics.ox.ac.uk.
Nicole C
NC
Robb
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1 3PU, UK.|Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Aartjan J W
AJ
Te Velthuis
Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany.
Ralph
R
Wieneke
Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany.
Robert
R
Tampé
Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Thorben
T
Cordes
Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands ervin.fodor@path.ox.ac.uk.
Ervin
E
Fodor
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1 3PU, UK kapanidis@physics.ox.ac.uk.
Achillefs N
AN
Kapanidis
D031541:Fluorescence Resonance Energy Transfer; D009980:Influenza A virus / Q000235:genetics*; D018613:Microscopy, Confocal; D008954:Models, Biological; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D061785:Transcription Initiation, Genetic; D014158:Transcription, Genetic; D014779:Virus Replication
D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase
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