Assembly, purification, and pre-steady-state kinetic analysis of active RNA-dependent RNA polymerase elongation complex.

ABSTRACT: NS5B is the RNA-dependent RNA polymerase responsible for replicating hepatitis C virus (HCV) genomic RNA. Despite more than a decade of work, the formation of a highly active NS5B polymerase·RNA complex suitable for mechanistic and structural studies has remained elusive. Here, we report that through a novel way of optimizing initiation conditions, we were able to generate a productive NS5B·primer·template elongation complex stalled after formation of a 9-nucleotide primer. In contrast to previous reports of very low proportions of active NS5B, we observed that under optimized conditions up to 65% of NS5B could be converted into active elongation complexes. The elongation complex was extremely stable, allowing purification away from excess nucleotide and abortive initiation products so tha

SUBMITTER: Jin Z 

PROVIDER: S-EPMC3323022 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature