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

Mitochondrial DNA is transcribed by single-subunit RNAPs (mtRNAP), which unlike their phage counterparts, depend on one or more transcription factors for promoter-specific transcription initiation. Much of our understanding of mitochondrial DNA transcription comes from studies of yeast (S. cerevisiae) and human mtRNAPs (2, 3, 5) . The yeast mtRNAP transcription initiation complex (y-mtIC) is comprised of the catalytic subunit RPO41 and a transcription factor MTF1. The human mtRNAP transcription initiation complex (h-mtIC) comprises of POLRMT and two transcription factors. The h-mtIC has been structurally characterized by crystallography (6) , but the structure was captured in an inactive fingersclenched state with a major part of the transcription bubble disordered. Hence, the structural basis for promoter melting, DNA scrunching, and transcription initiation remains largely unknown for the mtRNAPs. RPO41 (∆N100) and MTF1 were assembled on a pre-melted promoter (-21 to +12, 15S yeast mtDNA promoter; Fig. 1A ) to generate the yeast mitochondrial transcription preinitiation complex (y-mtPIC) (Fig. S1 ). The y-mtPIC was incubated with pppGpG RNA and a . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 non-hydrolysable UTPaS to generate the y-mtIC poised to incorporate the +3 NTP. Singleparticle cryo-EM data analysis of the quinary y-mtIC revealed a surprising coexistence of PIC and IC states in equilibrium (Fig. S2) . The y-mtIC structure had bound RNA and NTP, and y-mtPIC structure had no RNA or NTP. Another dataset collected from a PIC-only grid extends the resolution to 3.1 Å. Key steps guiding transcription initiation are revealed from the 3.1 Å y-mtPIC and 3.7 Å y-mtIC structures.