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

Biochemical studies show that the MTF1 C-tail plays an important role in template alignment, DNA scrunching, and triggering transition into elongation (15). The IC structure captures the entire C-tail in the active-site cavity and interacts with the template DNA, NTloop, and 5'-end of the RNA transcript. The C-tail base is stabilized by ICH, thumb helix, and a loop (521-526) of RPO41 (Fig. 3A ). The C-tail tip residue S340 is 4 Ã… away from the 5'end a-phosphate of pppGpG RNA. The main chain carbonyls of E338 and H339 hydrogen bond with the N1 and N6 atoms of the -2 template-base; similar interactions with N3 and N4 atoms of unmutated cytosine at -2 position are expected. The C-tail also stabilizes the scrunched NT-loop; M334-Y335 of C-tail stack against the looped-out +1 and +2 NT bases ( Fig. 3B ). Structural projection indicates that RNA synthesis will progressively push the Ctail out of its position in IC (Fig. 3C ), and at a critical length of RNA, the C-tail will be displaced out from the active-site cavity. Single-molecule FRET studies show that IC to EC transition completes at 8-mer RNA synthesis and C-tail deletion delays the transition (14, 15). Superposition of y-mtIC on POLRMT EC with 9-bp RNA:DNA (16) shows that C-tail . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 must exit for IC to EC transition (Fig. 3D ). We expect a similar role of C-tail in homologous h-mtIC (Fig. S6 ). Upon complete displacement of the C-tail, the MTF1-supporting hairpin will switch its role from guiding the C-tail in IC to supporting the upstream DNA in EC (16).