title: Bacteriophage phi 6 RNA-dependent RNA polymerase: molecular details of initiating nucleic acid synthesis without primer.
abstract: Like most RNA polymerases, the polymerase of double-strand RNA bacteriophage phi6 (phi6pol) is capable of primer-independent initiation. Based on the recently solved phi6pol initiation complex structure, a four-amino acid-long loop (amino acids 630-633) has been suggested to stabilize the first two incoming NTPs through stacking interactions with tyrosine, Tyr(630). A similar loop is also present in the hepatitis C virus polymerase, another enzyme capable of de novo initiation. Here, we use a series of phi6pol mutants to address the role of this element. As predicted, mutants at the Tyr(630) position are inefficient in initiation de novo. Unexpectedly, when the loop is disordered by changing Tyr(630)-Lys(631)-Trp(632) to GSG, phi6pol becomes a primer-dependent enzyme, either extending complementary oligonucleotide or, when the template 3' terminus can adopt a hairpin-like conformation, utilizing a "copy-back" initiation mechanism. In contrast to the wild-type phi6pol, the GSG mutant does not require high GTP concentration for its optimal activity. These findings suggest a general model for the initiation of de novo RNA synthesis.
authors.affiliation: Department of Biosciences and Institute of Biotechnology, P. O. Box 56, Viikinkaari 5, University of Helsinki, FIN-00014 Helsinki, Finland.
authors.forename: Minni R L
authors.initials: MR
authors.lastname: Laurila
authors.forename: Eugene V
authors.initials: EV
authors.lastname: Makeyev
authors.forename: Dennis H
authors.initials: DH
authors.lastname: Bamford
mesh_terms: D017126:Bacteriophage phi 6 / Q000201:enzymology*; D001483:Base Sequence; D018360:Crystallography, X-Ray; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000096:biosynthesis* / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
chemical_list: D014443:Tyrosine; D012313:RNA; D006160:Guanosine Triphosphate; D012324:RNA-Dependent RNA Polymerase; D012259:Ribonuclease, Pancreatic