Human mitochondrial RNA polymerase: evaluation of the single-nucleotide-addition cycle on synthetic RNA/DNA scaffolds.

ABSTRACT: The human mitochondrial RNA polymerase (h-mtRNAP) serves as both the transcriptase for expression and the primase for replication of mitochondrial DNA. As such, the enzyme is of fundamental importance to cellular energy metabolism, and defects in its function may be related to human disease states. Here we describe in vitro analysis of the h-mtRNAP kinetic mechanism for single, correct nucleotide incorporation. This was made possible by the development of efficient methods for expression and purification of h-mtRNAP using a bacterial system and by utilization of assays that rely on simple, synthetic RNA/DNA scaffolds without the need for mitochondrial transcription accessory proteins. We find that h-mtRNAP accomplishes single-nucleotide incorporation by using the same core steps, including

SUBMITTER: Smidansky ED 

PROVIDER: S-EPMC3698222 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature