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Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA

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Robb, Nicole C., te Velthuis, Aartjan J. W., Wieneke, Ralph, Tampé, Robert, Cordes, Thorben, Fodor, Ervin and Kapanidis, Achillefs N. (2016) Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA. Nucleic Acids Research, 44 (21). pp. 10304-10315. doi:10.1093/nar/gkw884

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Official URL: http://dx.doi.org/10.1093/nar/gkw884

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Abstract

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.

Item Type: Journal Article
Subjects: Q Science > QC Physics
Q Science > QP Physiology
Q Science > QR Microbiology
Divisions: Faculty of Medicine > Warwick Medical School > Biomedical Sciences > Cell & Developmental Biology
Faculty of Medicine > Warwick Medical School > Biomedical Sciences
Faculty of Medicine > Warwick Medical School
Library of Congress Subject Headings (LCSH): Influenza viruses, Influenza viruses -- Reproduction , RNA, Energy transfer, Molecular dynamics
Journal or Publication Title: Nucleic Acids Research
Publisher: Oxford University Press
ISSN: 0305-1048
Official Date: December 2016
Dates:
DateEvent
December 2016Published
30 September 2016Available
1 December 2016Accepted
Date of first compliant deposit: 28 July 2020
Volume: 44
Number: 21
Page Range: pp. 10304-10315
DOI: 10.1093/nar/gkw884
Status: Peer Reviewed
Publication Status: Published
Access rights to Published version: Open Access
RIOXX Funder/Project Grant:
Project/Grant IDRIOXX Funder NameFunder ID
110164/Z/15/ZWellcome Trusthttp://dx.doi.org/10.13039/100010269
098721/Z/12/ZWellcome Trusthttp://dx.doi.org/10.13039/100010269
BB/J001694/1[BBSRC] Biotechnology and Biological Sciences Research Councilhttp://dx.doi.org/10.13039/501100000268
MR/N010744/1[MRC] Medical Research Councilhttp://dx.doi.org/10.13039/501100000265
MR/K000241/1[MRC] Medical Research Councilhttp://dx.doi.org/10.13039/501100000265
825.11.029[NWO] Nederlandse Organisatie voor Wetenschappelijk Onderzoekhttp://dx.doi.org/10.13039/501100003246
Kemp postdoctoral fellowshipLincoln College, University of Oxfordhttp://dx.doi.org/10.13039/100010371
EXC115Deutsche Stiftung Friedensforschunghttp://dx.doi.org/10.13039/501100007450
SPP 1623Deutsche Stiftung Friedensforschunghttp://dx.doi.org/10.13039/501100007450
SFB 902 Deutsche Stiftung Friedensforschunghttp://dx.doi.org/10.13039/501100007450
ERC-STG-638536European Research Councilhttp://dx.doi.org/10.13039/501100000781
personal start-up grantRijksuniversiteit Groningenhttp://dx.doi.org/10.13039/501100001721

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