Transcriptional Fidelity of Mitochondrial RNA Polymerase RpoTm from Arabidopsis thaliana

Abstract

Fidelity of RNA synthesis is essential for the faithful transfer of information from DNA to RNA. A comprehensive analysis of the nucleotide selectivity by the mitochondrial RNA polymerase (RNAP) RpoTm, from Arabidopsis thaliana, has been carried out. The kinetic parameters for the incorporation of cognate, noncognate, and oxidized bases have been determined. The results establish high fidelity of mitochondrial transcription resembling those of replicative polymerases in the absence of repair. In addition, RpoTm incorporates oxidized nucleotides with similar efficiency compared with mismatches and is capable of extending the RNA beyond the insertion of the oxidized base. Furthermore, lesion bypass study on RpoTm demonstrates that the enzyme bypasses 8-oxo-guanine by insertion of adenine leading to C to A mutations in RNA. Homology modeling of RpoTm elongation complex allows delineation of the residues necessary for stabilizing the incoming NTP substrate and for posing the template nucleotide residue. Substitution of these residues leads to compromise in the activity of the enzyme corroborating their importance in RNA synthesis. Comparison of the data with T7 RNAPs indicates that low efficiency of misincorporation is a universal strategy used by single-subunit RNAPs for maintaining high fidelity in the absence of proofreading and repair activity in mitochondria.

Keywords: 8-Oxo-guanine; Arabidopsis thaliana; Mitochondrial RNAP; ROS; Transcription.