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J. Biol. Chem., Vol. 277, Issue 19, 17117-17124, May 10, 2002

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Bacteriophage 6 RNA-dependent RNA Polymerase
MOLECULAR DETAILS OF INITIATING NUCLEIC ACID SYNTHESIS WITHOUT PRIMER^*

Minni R. L. Laurila, Eugene V. Makeyev, and Dennis H. Bamford

From the Department of Biosciences and Institute of Biotechnology, P. O. Box 56, Viikinkaari 5, University of Helsinki, FIN-00014 Helsinki, Finland

Like most RNA polymerases, the polymerase of double-strand RNA bacteriophage 6 (6pol) is capable of primer-independent initiation. Based on the recently solved 6pol 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 6pol 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, 6pol 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 6pol, 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.

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