Cryo-EM structures reveal transcription initiation steps by yeast mitochondrial RNA polymerase

Brent De Wijngaert; Shemaila Sultana; Chhaya Dharia; Hans Vanbuel; Jiayu Shen; Daniel vasilchuk; Sergio E Martinez; Eaazhisai Kandiah; Smita S Patel; Kalyan Das

The PIC and IC structures provide a basis for understanding the mechanism of abortive synthesis. Abortive synthesis is observed in all DNA-dependent RNAPs during transcription initiation, and y-mtRNAP generates large amounts of 2-mer and 3-mer abortive products compared to 4-to 6-mer abortives ( The y-mtIC has captured the incoming NTP in a catalytic-competent state poised for incorporation (Fig. 4A) , The NTP and the DNA:RNA duplex make extensive interactions with RPO41 in the active site, which is highly conserved in mitochondrial RNAPs ( Fig. S7 and S8). The structure of y-mtIC permits reliable modeling of antiviral nucleosides/nucleotides for cytotoxicity prediction. Nucleos(t)ide analogs are widely used . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.13.038620 doi: bioRxiv preprint to treat viral infections and can cause cytotoxicity by binding to cellular RNAP and mitochondrial POLRMT. Remdesivir is a nucleotide analog with broad antiviral profile (20) including treatment of SARS-CoV-2 (COVID-19) infection. Modeling of remdesivirdiphosphate into the NTP-binding pocket of mtRNAP reveals that the characteristic 1'cyano group of remdesivir clashes with the conserved H1125 in POLRMT (Fig. 4B) . Thus, remdesivir is expected to have low cytotoxicity, consistent with its low incorporation efficiency by POLRMT (20, 21). Thus, the platform provides a framework for testing mitochondrial toxicity of nucleoside analogs.