The Dynamic Landscape of Transcription Initiation in Yeast Mitochondria

Abstract

12Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is 13 crucial for regulating cellular energy metabolism. Studies of bacteriophage and bacterial 14 systems have revealed that transcription occurs through a series of conformational transitions 15 during the initiation and elongation stages; however, how the conformational dynamics 16 progress throughout these stages remains unknown. Here, we used single-molecule 17 fluorescence resonance energy transfer techniques to examine the conformational dynamics 18 of the two-component transcription system of yeast mitochondria with single-base resolution. 19We show that, unlike its single-component homologue in bacteriophages, the yeast 20 mitochondrial transcription initiation complex dynamically transitions between closed, open, 21 and scrunched conformations throughout the initiation stage, and then makes a sharp 22 irreversible transition to an unbent conformation by promoter release at position +8. 23Remarkably, stalling the initiation complex revealed unscrunching dynamics without 24 dissociating the RNA transcript, manifesting the existence of backtracking transitions with 1 possible regulatory roles. The dynamic landscape of transcription initiation revealed here 2 suggests a kinetically driven regulation of mitochondrial transcription. 3 4 stabilizing open promoter regions 6,[16][17][18] . Similarly, human mitochondrial transcription 1 requires the initiation factors TFAM and TFB2M [19][20][21] . Mtf1 is structurally and functionally 2 homologous to TFB2M 19,22 , and is functionally similar to the bacterial sigma factor 23-25 . To 3 regulate transcription efficiency at various promoters, these transcription systems have 4 developed a set of molecular mechanisms, in which they share several key features. Both 5 bacteriophage and bacterial RNAPs show scrunching of the downstream DNA into the active 6 site as the RNA-DNA hybrid and the transcription bubble grow during initiation. After a 7 stable full-length RNA-DNA hybrid is formed, the RNAP releases upstream promoter 8 contacts and the initiation bubble collapses to drive the transition into elongation 26-32 . A 9 similar mechanism is also thought to exist in higher organisms 33,34 . More recently, branching 10 between competing pathways and pausing during initiation have been observed in both 11 bacteriophages and bacteria and may play crucial roles in regulating transcription 12 activity 1,3,4,35 . 13 In yeast mitochondria, transcription initiates with the assembly of Rpo41 and Mtf1 at 14 conserved promoter sequences located at positions −8 to +1 relative to the transcription start 15 site. The 2-aminopurine (2AP) fluorescence and protein-DNA crosslinking experiments have 16shown that Mtf1 facilitates promoter melting at positions −4 to +2 by trapping the non-17 template strand 24,25,36 . The structures of T7 and human mitochondrial RNAPs show that the 18 promoter DNA is severely bent around the start-site in the open complex 37,38 . Single-19 molecule studies of ...

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

“…This observation is consistent with single-molecule fluorescence studies showing that T7 and bacterial RNAPs remain stably bound to the promoter during abortive synthesis (Koh et al, 2018;Revyakin et al, 2006). Continuous scrunching/unscrunching, which we infer is occurring on the basis of the coexistence of both states in solution, is consistent with a recent single-molecule FRET study of y-mtRNAP where dynamic switching between scrunched and unscrunched states was observed throughout transcription initiation (Sohn et al, 2020). The dynamic equilibrium between IC and PmIC states in our study suggests that in an abortive event, the scrunched IC state switches to the unscrunched PmIC state, which will then rebind the NTPs to start a fresh cycle of RNA synthesis.…”

“…The co-existence of PmIC and IC states suggests a dynamic equilibrium between the two states. A recent single-molecule FRET study of transcription initiation observed dynamic equilibrium among intermediate IC states of y-mtRNAP (Sohn et al, 2020). From the cryo-EM dataset, the final density maps for y-mtRNAP IC and y-mtRNAP PmIC structures were calculated at 3.7 Å and 3.5 Å resolution, respectively (Table 1).…”

“…DNA scrunching has been proposed as an underlying consequence of the mechanism of transcription initiation by DNAdependent RNAPs, including y-mtRNAP (Cheetham and Steitz, 1999;Kapanidis et al, 2006;Sohn et al, 2020;Tang et al, 2008). However, the conformation of the scrunched DNA has not been captured in any of the high-resolution structures and our y-mtRNAP IC structure is the first to capture the scrunched DNA conformation (Figure 3C).…”

“…At a critical length of RNA, the C-tail will be displaced out of the active-site cavity. Single-molecule FRET studies of the y-mtRNAP IC show that the transition from initiation to an early elongation state completes at 8-mer RNA synthesis and MTF1 C-tail deletion delays this transition (Basu et al, 2020;Sohn et al, 2020). The superposition of y-mtRNAP IC and h-mtRNAP IC structures reveals that h-mtRNAP and TFB2M superimpose reasonably well on y-mtRNAP and MTF1, respectively (Figures S6B and S6C), and their respective promoter DNAs are aligned as well (Figure S6D).…”

“…However, the upstream promoter DNA remains stably bound to the RNAP throughout initiation; therefore, the transcription bubble progressively gets larger with RNA synthesis. The expanding bubble is accommodated in the active-site cavity through DNA scrunching, which is a general mechanism observed in both multi-and single-subunit RNAPs (Kapanidis et al, 2006;Tang et al, 2008), including y-mtRNAP (Sohn et al, 2020). Despite its widespread acceptance as a mechanism for transcription initiation, the scrunched DNA conformation has not been visualized in any system.…”