title: Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage phi6.
abstract: In nature, synthesis of both minus- and plus-sense RNA strands of all the known double-stranded RNA viruses occurs in the interior of a large protein assembly referred to as the polymerase complex. In addition to other proteins, the complex contains a putative polymerase possessing characteristic sequence motifs. However, none of the previous studies has shown template-dependent RNA synthesis directly with an isolated putative polymerase protein. In this report, recombinant protein P2 of double-stranded RNA bacteriophage phi6 was purified and demonstrated in an in vitro enzymatic assay to act as the replicase. The enzyme efficiently utilizes phage-specific, positive-sense RNA substrates to produce double-stranded RNA molecules, which are formed by newly synthesized, full-length minus-strands base paired with the plus-strand templates. P2-catalyzed replication is also shown to be very effective with a broad range of heterologous single-stranded RNA templates. The importance and implications of these results are discussed.
authors.affiliation: Institute of Biotechnology and Department of Biosciences, PO Box 56, Viikinkaari 5, FIN-00014, University of Helsinki, Finland.
authors.forename: E V
authors.initials: EV
authors.lastname: Makeyev
authors.forename: D H
authors.initials: DH
authors.lastname: Bamford
mesh_terms: D000595:Amino Acid Sequence; D017126:Bacteriophage phi 6 / Q000201:enzymology*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli; D008969:Molecular Sequence Data; D012330:RNA, Double-Stranded / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013698:Templates, Genetic
chemical_list: D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; C023140:DNA replicase; D012324:RNA-Dependent RNA Polymerase; D004259:DNA-Directed DNA Polymerase; D012260:Ribonucleases