Identification of the RNA polymerase-binding site on genome RNA of influenza virus.
The binding sites for influenza viral RNA polymerase on genome RNA segments were investigated. Ribonucleoprotein (RNP) cores containing the RNA polymerase were isolated from detergent-treated virions by glycerol gradient centrifugation. On ApG-primed in vitro transcription by the isolated RNP cores, different levels of RNA transcripts were synthesized for the eight RNP cores, suggesting an uneven distribution of the RNA polymerase. 3'-Terminal labeling of the RNP cores with the use of [32P]pCp and T4-RNA ligase indicated a reciprocal correlation between the levels of the RNA-3' label and RNA synthesis. Centrifugation of detergent-treated virions in a double gradient of cesium trifluoroacetate (or cesium chloride) and glycerol yielded RNA polymerase-RNA complexes devoid of NP, the major RNA-bound protein, but the pattern of RNA-3' labeling remained virtually unaffected. All these observations together indicated that the RNA polymerase is associated near the 3' termini of some viral RNA segments, thereby preventing the in vitro labeling of the RNA-3' ends. The results of foot-printing experiments using RNase V1 and RNase T2 were in agreement with this model.
Honda
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Department of Molecular Genetics, National Institute of Genetics, Shizuoka.
Uéda
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Nagata
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Ishihama
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D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D003565:Cytidine Diphosphate / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015226:Dinucleoside Phosphates; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000378:metabolism; D005814:Genes, Viral; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D009975:Orthomyxoviridae / Q000201:enzymology / Q000235:genetics*; D012367:RNA, Viral / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D014158:Transcription, Genetic
D015226:Dinucleoside Phosphates; D009711:Nucleotides; D012367:RNA, Viral; D012261:Ribonucleoproteins; D006151:Guanosine; C032883:cytidine 3',5'-diphosphate; C031924:adenylyl-(3'-5')-guanosine; D000249:Adenosine Monophosphate; D003565:Cytidine Diphosphate; D012321:DNA-Directed RNA Polymerases; D004722:Endoribonucleases; C032825:ribonuclease V(1); C022543:ribonuclease T(2)