Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase

Abstract

Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production yet, understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We report structures of two transcription initiation intermediate states of yeast mtRNAP that explain promoter melting, template alignment, DNA scrunching, abortive synthesis, and transition into elongation. In partially-melted initiation complex (PmIC), transcription factor MTF1 makes base-specific interactions with flipped non-template (NT) -4 to -1 nucleotides 'AAGT' of the DNA promoter. In the initiation complex (IC), the template in the expanded 7-mer bubble positions the RNA and NTP analog, UTP S, while NT scrunches into an NT-loop. The scrunched NT-loop is stabilized by centrally positioned MTF1 C-tail. The IC and PmIC states coexist in solution revealing a dynamic equilibrium between two functional states. Frequent scrunching/unscruching transitions, and the imminent steric clashes of inflating NT-loop and growing RNA:DNA with the C-tail explain abortive synthesis and transition into elongation.

The dynamic landscape of transcription initiation in yeast mitochondria

“…The open promoter structure with human mitochondrial RNAP (PDB: 6ERP) 45 exhibits a sharply bent promoter DNA, consistent with the increase in FRET level upon promoter opening. Recently revealed cryo-EM structure of Rpo41 and Mtf1 is consistent with the smFRET results here 39 . The structure of T7 RNAP with 6 bp RNA:DNA (PDB: 3E2E) 47 shows a more sharply bent DNA, consistent with the elevated FRET level we observed in IC6.…”

“…Mtf1 on its own does not bind DNA because the C-terminal tail autoinhibits DNA binding 37 , but in complex with Rpo41, Mtf1 drives promoter melting by trapping the non-template strand of the initiation bubble at positions −4 to +2 (refs. 26,27,38,39 ). Biochemical and recent structural studies of yeast mitochondrial RNAP show that the promoter DNA is severely bent around the transcription start site in the open complex 39,40 .…”

“…26,27,38,39 ). Biochemical and recent structural studies of yeast mitochondrial RNAP show that the promoter DNA is severely bent around the transcription start site in the open complex 39,40 . Single-molecule studies show that the open promoter dynamically switches between bent and unbent conformations prior to binding the initiating nucleotides 4 .…”

Yeast mtDNA transcription initiation in single nucleotide addition steps

“…1b, c). Together with the published PmIC (PDB: 6YMV) and IC3 (PDB: 6YMW) structures 19 , the four new IC structures help visualize the progression of transcription initiation (Fig. 1d, e, f).…”

Yeast mtDNA transcription initiation in single nucleotide addition steps

“…The y-mtRNAP requires one factor, MTF1, and h-mtRNAP requires two factors, TFAM and TFB2M, for promoterspecific transcription initiation 10,11,[16][17][18] ; T7 RNAP does not require any factor. Recently, we published high-resolution structures of y-mtRNAP:MTF1 with a DNA promoter in the partiallymelted initiation complex (PmIC) and a transcribing IC3 states 19 . These structures resolved the complete transcription bubble, which could not be traced in the IC structures of h-mtRNAP 20 or T7 RNAP 21 .…”