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 stru...
| Main Authors: | , , , , , , |
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| Format: | Journal article |
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Oxford University Press
2016
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| _version_ | 1826294320218505216 |
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| author | Robb, N te Velthuis, A Wieneke, R Tampe, R Cordes, T Fodor, E Kapanidis, A |
| author_facet | Robb, N te Velthuis, A Wieneke, R Tampe, R Cordes, T Fodor, E Kapanidis, A |
| author_sort | Robb, N |
| collection | OXFORD |
| description | 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 provides 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. |
| first_indexed | 2024-03-07T03:43:49Z |
| format | Journal article |
| id | oxford-uuid:bec32d72-4423-47e0-8c28-42d4075ad31b |
| institution | University of Oxford |
| last_indexed | 2024-03-07T03:43:49Z |
| publishDate | 2016 |
| publisher | Oxford University Press |
| record_format | dspace |
| spelling | oxford-uuid:bec32d72-4423-47e0-8c28-42d4075ad31b2022-03-27T05:42:27ZSingle-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNAJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:bec32d72-4423-47e0-8c28-42d4075ad31bSymplectic Elements at OxfordOxford University Press2016Robb, Nte Velthuis, AWieneke, RTampe, RCordes, TFodor, EKapanidis, AInfluenza 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 provides 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. |
| spellingShingle | Robb, N te Velthuis, A Wieneke, R Tampe, R Cordes, T Fodor, E Kapanidis, A Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA |
| title | Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus
promoter RNA |
| title_full | Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus
promoter RNA |
| title_fullStr | Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus
promoter RNA |
| title_full_unstemmed | Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus
promoter RNA |
| title_short | Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus
promoter RNA |
| title_sort | single molecule fret reveals the pre initiation and initiation conformations of influenza virus promoter rna |
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