Transcriptional fidelities of human mitochondrial POLRMT, yeast mitochondrial Rpo41, and phage T7 single-subunit RNA polymerases

Abstract Single-subunit RNA polymerases (RNAPs) are present in phage T7 and in mitochondria of all eukaryotes. This RNAP class plays important roles in biotechnology and cellular energy production, but we know little about its fidelity and error rates. Herein, we report the error rates of three single-subunit RNAPs measured from the catalytic efficiencies of correct and all possible incorrect nucleotides. The average error rates of T7 RNAP (2 × 10−6), yeast mitochondrial Rpo41 (6 × 10−6), and human mitochondrial POLRMT (RNA polymerase mitochondrial) (2 × 10−5) indicate high accuracy/fidelity of RNA synthesis resembling those of replicative DNA polymerases. All three RNAPs exhibit a distinctly high propensity for GTP misincorporation opposite dT, predicting frequent A→G errors in RNA with rates of ∼10−4. The A→C, G→A, A→U, C→U, G→U, U→C, and U→G errors mostly due to pyrimidine–purine mismatches were relatively frequent (10−5–10−6), whereas C→G, U→A, G→C, and C→A errors from purine–purine and pyrimidine–pyrimidine mismatches were rare (10−7–10−10). POLRMT also shows a high C→A error rate on 8-oxo-dG templates (∼10−4). Strikingly, POLRMT shows a high mutagenic bypass rate, which is exacerbated by TEFM (transcription elongation factor mitochondrial). The lifetime of POLRMT on terminally mismatched elongation substrate is increased in the presence of TEFM, which allows POLRMT to efficiently bypass the error and continue with transcription. This investigation of nucleotide selectivity on normal and oxidatively damaged DNA by three single-subunit RNAPs provides the basic information to understand the error rates in mitochondria and, in the case of T7 RNAP, to assess the quality of in vitro transcribed RNAs.