TY - JOUR
T1 - Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA
AU - Robb, Nicole C.
AU - Te Velthuis, Aartjan J.W.
AU - Wieneke, Ralph
AU - Tamṕe, Robert
AU - Cordes, Thorben
AU - Fodor, Ervin
AU - Kapanidis, Achillefs N.
N1 - Publisher Copyright:
© 2016 The Author(s).
PY - 2016/12
Y1 - 2016/12
N2 - Influenza viruses have a segmented viralRNA(vRNA) genome, which is replicated by the viral RNAdependent 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.
AB - Influenza viruses have a segmented viralRNA(vRNA) genome, which is replicated by the viral RNAdependent 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.
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U2 - 10.1093/nar/gkw884
DO - 10.1093/nar/gkw884
M3 - Article
C2 - 27694620
AN - SCOPUS:85016161732
SN - 0305-1048
VL - 44
SP - 10304
EP - 10315
JO - Nucleic acids research
JF - Nucleic acids research
IS - 21
ER -