Bacteriophage φ6 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 φ6 (φ6pol) is capable of primer-independent initiation. Based on the recently solved φ6pol 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, Tyr630. 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 φ6pol mutants to address the role of this element. As predicted, mutants at the Tyr630 position are inefficient in initiation de novo. Unexpectedly, when the loop is disordered by changing Tyr630-Lys631-Trp632 to GSG, φ6pol 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 φ6pol, the GSG mutant does not require high GTP concentration for its optimal activity. These findings suggest a general model for the initiation ofde novo RNA synthesis.