Mutational analysis of the role of nucleoside triphosphatase P4 in the assembly of the RNA polymerase complex of bacteriophage phi6.
Bacteriophage phi6 is a complex enveloped double-stranded RNA virus with a segmented genome and replication strategy quite similar to that of the Reoviridae. An in vitro packaging and replication system using purified components is available. The positive-polarity genomic segments are translocated into a preformed polymerase complex (procapsid) particle. This particle is composed of four proteins: the shell-forming protein P1, the RNA polymerase P2, and two proteins active in packaging. Protein P7 is involved in stable packaging, and protein P4 is a homomultimeric potent nucleoside triphosphatase that provides the energy for the RNA translocation event. In this investigation, we used mutational analysis to study P4 multimerization and assembly. P4 is assembled onto a preformed particle containing proteins P2 and P7 in addition to P1. Only simultaneous production of P1 and P4 in the same cell leads to P4 assembly on P1 alone, whereas the P1 shell is incompetent for accepting P4 if produced separately. The C-terminal part of P4 is essential for particle assembly but not for multimerization or enzymatic activity. Altering the P4 nucleoside triphosphate binding site destroys the ability to form multimers.
Paatero
A O
AO
Department of Biosciences, Biocenter, FIN-00014, University of Helsinki, Finland.
Mindich
L
L
Bamford
D H
DH
D017126:Bacteriophage phi 6 / Q000201:enzymology* / Q000235:genetics* / Q000648:ultrastructure; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005814:Genes, Viral; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D004279:DNA, Viral; D014764:Viral Proteins; C080701:protein P4 (Pseudomonas); D012321:DNA-Directed RNA Polymerases; D010744:Phosphoric Monoester Hydrolases