PMID:11877396

Citation	Laurila, MR, Makeyev, EV and Bamford, DH (2002) Bacteriophage phi 6 RNA-dependent RNA polymerase: molecular details of initiating nucleic acid synthesis without primer. J. Biol. Chem. 277:17117-24
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.
Links	PubMed Online version:10.1074/jbc.M111220200
Keywords	Bacteriophage phi 6/enzymology; Base Sequence; Crystallography, X-Ray; Dimerization; Dose-Response Relationship, Drug; Electrophoresis, Agar Gel; Guanosine Triphosphate/metabolism; Kinetics; Models, Biological; Models, Molecular; Molecular Sequence Data; Mutation; Nucleic Acid Conformation; Plasmids/metabolism; Protein Binding; Protein Conformation; Protein Structure, Tertiary; RNA/biosynthesis; RNA/metabolism; RNA Replicase/chemistry; Ribonuclease, Pancreatic/metabolism; Tyrosine/chemistry
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPPH6:RDRP		GO:0003968: RNA-directed RNA polymerase activity	ECO:0000314:		F		Figure 2B shows that wild type phi6 RdRp was able to utilize the ssRNA substrate and form dsRNA product.	complete CACAO 5986

References

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