title: Structure-function relationship of the influenza virus RNA polymerase: primer-binding site on the PB1 subunit.
abstract: Influenza virus RNA polymerase is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the viral RNA genome. The catalytic site for RNA polymerization is located on the PB1 subunit. To identify the primer ATP-binding site, we have employed a highly selective cross-linking technique: three structurally diverse ATP analogues with reactive groups on their phosphate moieties were first cross-linked to the viral RNA polymerase, and the cross-linked nucleotides were then elongated to dinucleotides by adding the second substrate [alpha-(32)P]GTP. Only the ATP analogues tethered to the primer-binding site could be elongated to radioactive AG dinucleotides. Using this catalytically competent cross-linking procedure, the PB1 subunit was found to be the only labeled subunit. Limited proteolysis of the labeled PB1 by V8 protease revealed the segment between amino acids 179 and 297 as the only bearer of the radioactive label. Thus, we concluded that this region of PB1 faces the 5' end of the primer nucleotide. In support of this prediction, the cross-linked dinucleotides were further elongated up to 8-10 nucleotides in length upon addition of all four substrates. This result suggests that the substantial mass of RNA can be accommodated between the binding site for the primer (and nascent RNA) and the catalytic center of RNA polymerization.
authors.affiliation: Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.
authors.forename: Dmitry M
authors.initials: DM
authors.lastname: Kolpashchikov
authors.forename: Ayae
authors.initials: A
authors.lastname: Honda
authors.forename: Akira
authors.initials: A
authors.lastname: Ishihama
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D018387:Codon, Initiator / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D009975:Orthomyxoviridae / Q000201:enzymology*; D010441:Peptide Chain Elongation, Translational; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis; D012261:Ribonucleoproteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D018387:Codon, Initiator; D003432:Cross-Linking Reagents; D017931:DNA Primers; D021122:Protein Subunits; D012367:RNA, Viral; D012261:Ribonucleoproteins; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; C034220:cyclic adenosine-5'-trimetaphosphate; D000242:Cyclic AMP; D012321:DNA-Directed RNA Polymerases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase