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

. 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.13.038620 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the .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 1QLN) shows that the promoter DNA template in y-mtIC (pink) is bent sharply at the active site and after 4 nucleotides that is analogous to the template track in T7 RNAP IC (gray); however, the bound UTPaS captures y-mtIC in the catalytic mode for nucleotide incorporation whereas, the T7 RNAP IC structure represents the post-translocated state with no bound NTP. (C) The comparison also shows that the NTP-binding pocket undergoes a conformational change. The conserved Y639 on the O helix of T7 RNAP must shift to the position that Y1022 of RPO41 takes to accommodate an NTP. (D) Superposition of y-mtIC on h-mtIC structure (PDB Id. 6EQR) shows that the Y-helix of POLRMT clashes with the template strand and the state observed in the h-mtIC structure would not position the template in a conformation that is compatible for RNA/NTP-binding. The h-mtIC structure represents an inactive-clenched state. The Y-helix (1023-1041 of RPO41) is critical for downstream DNA unwinding.