Publication

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

Robb, N. C., te Velthuis, A. J. W., Wieneke, R., Tampe, R., Cordes, T., Fodor, E. & Kapanidis, A. N., Dec-2016, In : Nucleic Acids Research. 44, 21, p. 10304-10315 12 p.

Research output: Contribution to journalArticleAcademicpeer-review

  • Nicole C. Robb
  • Aartjan J. W. te Velthuis
  • Ralph Wieneke
  • Robert Tampe
  • Thorben Cordes
  • Ervin Fodor
  • Achillefs N. Kapanidis

Influenza viruses have a segmented viralRNA(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 Forster 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.

Original languageEnglish
Pages (from-to)10304-10315
Number of pages12
JournalNucleic Acids Research
Volume44
Issue number21
Publication statusPublished - Dec-2016

    Keywords

  • ALTERNATING-LASER EXCITATION, RESONANCE ENERGY-TRANSFER, DNA-POLYMERASE-I, TAGGED PROTEINS, VIRAL-RNA, C VIRUS, TRANSCRIPTION, REPLICATION, CRNA, COMPLEXES

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