key: cord-0325553-ex3zlq38
authors: De Wijngaert, Brent; Sultana, Shemaila; Dharia, Chhaya; Vanbuel, Hans; Shen, Jiayu; Vasilchuk, Daniel; Martinez, Sergio E.; Kandiah, Eaazhisai; Patel, Smita S.; Das, Kalyan
title: Cryo-EM structures reveal transcription initiation steps by yeast mitochondrial RNA polymerase
date: 2020-04-14
journal: bioRxiv
DOI: 10.1101/2020.04.13.038620
sha: 47fcf7531d4b5b06799ddcc4e1efcb886526820b
doc_id: 325553
cord_uid: ex3zlq38

Cryo-EM structures of transcription pre-initiation complex (PIC) and initiation complex (IC) of yeast mitochondrial RNA polymerase show fully resolved transcription bubbles and explain promoter melting, template alignment, DNA scrunching, transition into elongation, and abortive synthesis. Promoter melting initiates in PIC with MTF1 trapping the −4 to −2 non-template (NT) bases in its NT-groove. Transition to IC is marked by a large-scale movement that aligns the template with RNA at the active site. RNA synthesis scrunches the NT strand into an NT-loop, which interacts with centrally positioned MTF1 C-tail. Steric clashes of the C-tail with RNA:DNA and NT-loop, and dynamic scrunching-unscrunching of DNA explain abortive synthesis and transition into elongation. Capturing the catalytically active IC-state with UTPαS poised for incorporation enables modeling toxicity of antiviral nucleosides/nucleotides.

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 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.

In y-mtPIC structure ( Fig. 1B & C) , the MTF1 is traced from 2 to 336 out of 341 amino acid residues, RPO41 is traced from 386 to the end residue 1351 with few disordered regions, and unambiguously traced DNA (Fig. S3A) . A stable transcription core is composed of RPO41, MTF1, and transcription bubble (Fig. 1C ). RPO41 interacts with MTF1 at multiple locations. Two RPO41 b-hairpins -the intercalating hairpin (ICH) and the MTF1supporting hairpin (K613-P632) form a crescent-shaped platform that accommodates the C-terminal domain of MTF1 (252-325) (Fig. 1D) . The N-terminal domain of MTF1 contacts the tip of the RPO41 thumb helix (Fig. 1E) ; biochemically, we show the interaction stabilizes RPO41-MTF1 complex ( Fig. S3C -D) (7) . The MTF1-supporting hairpin also guides the MTF C-tail (326-341) towards the active site (Fig. 1F ); the C-tail is disordered in free MTF1 (8) .

The PIC structure has not been observed previously; thus, it provides new insights into the mechanism of promoter melting. The y-mtPIC structure suggests that RPO41 and MTF1 initiate the promoter melting by creating a 4-nt transcription bubble from -4 to -1. We provided a -4 to +2 pre-melted promoter; however, the +1 and +2 nucleotides assume a duplex-like DNA conformation in PIC albeit lacking canonical base-pairing. DNA melting is driven by sequence-specific interactions of the NT strand with the NT-groove that lies at the interface of N-and C-terminal domains of MTF1 (residues 103-105, 144-148, and 190-192) . The -4 to -2 AAG bases in the NT strand are flipped towards NT-groove (Fig. 1G ). The -2 guanine base is sandwiched between the aromatic side chains of Y103 and W105, and all N and O atoms of the base, except N7, are engaged in complementary hydrogen bond interactions (Fig. 1H) ; mutation of -2 guanine severely impair promoter melting (9) . The -1 NT base stacks with Y103 with no base-specific interaction. The -3 and -4 AA bases are The quinary y-mtIC has a promoter designed to bind a 2-mer RNA and incorporate a third nucleotide ( Fig. 2A) . The 3.7 Å density map of IC locates many previously uncharacterized structural elements, including the C-tail of MTF1 and the scrunched DNA ( Fig. 2B-C) , and reveals the mechanism of template alignment and RNA synthesis during initiation.

Comparison of PIC and IC shows little shift of upstream promoter DNA and interacting structural elements (including ICH, specificity loop, and thumb of RPO41 and NT-groove of MTF1) during PIC to IC transition. In contrast, the downstream DNA and interacting Cterminal domain of RPO41 undergo large conformational changes including fingers closing (Movie S1). During the transition, the upstream DNA from position -1 onward is locked in the MTF1 NT-groove while the template strand undergoes a large conformational switching to align with the 2-mer RNA and the incoming UTPaS at the active site ( Fig. 2C; Movie S2). These unsynchronized events at two ends of the transcription bubble scrunches the NT strand into an NT-loop (Movie S3). DNA scrunching has been proposed in multi-and single-subunit RNAPs including y-mtRNAP (11) (12) (13) (14) . Our y-mtIC structure is first to capture the scrunched conformation (Fig. 2C) . The scrunched NT-loop is stabilized by ICH (H641, N642), thumb (R780 and K787), and MTF1 C-tail (M334-Y335). The looping of NT strand alters the downstream DNA track and bends it from ~60° to ~120° with respect to the upstream DNA; thus, transforming a V-shaped DNA in PIC to an U-shape in IC (Fig. S5 ).

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 Å 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 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).

The PIC and IC structures provide a basis for understanding the mechanism of abortive synthesis. Abortive synthesis is observed in all DNA-dependent RNAPs during transcription initiation, and y-mtRNAP generates large amounts of 2-mer and 3-mer abortive products compared to 4-to 6-mer abortives ( The y-mtIC has captured the incoming NTP in a catalytic-competent state poised for incorporation (Fig. 4A) , The NTP and the DNA:RNA duplex make extensive interactions with RPO41 in the active site, which is highly conserved in mitochondrial RNAPs ( Fig. S7 and S8). The structure of y-mtIC permits reliable modeling of antiviral nucleosides/nucleotides for cytotoxicity prediction. Nucleos(t)ide analogs are widely used to treat viral infections and can cause cytotoxicity by binding to cellular RNAP and mitochondrial POLRMT. Remdesivir is a nucleotide analog with broad antiviral profile (20) including treatment of SARS-CoV-2 (COVID-19) infection. Modeling of remdesivirdiphosphate into the NTP-binding pocket of mtRNAP reveals that the characteristic 1'cyano group of remdesivir clashes with the conserved H1125 in POLRMT (Fig. 4B) . Thus, remdesivir is expected to have low cytotoxicity, consistent with its low incorporation efficiency by POLRMT (20, 21). Thus, the platform provides a framework for testing mitochondrial toxicity of nucleoside analogs.

G. Q. Tang The coordinates and density maps for y-mtPIC and y-mtIC structures were deposited under PDB accession numbers 6YMV and 6YMW and EMBD Ids. EMD-10845 and EMD-10846, respectively. All data is available in the main text or the supplementary materials. All data, code, and materials used in the analysis will be available for purposes of reproducing or extending the analysis. (B) Superposition of y-mtPIC structure (black non-template; gray template) on y-mtIC structure (cyan non-template; pink template) shows the bending of downstream DNA while the upstream DNA in both structures are aligned. The angle between the upstream and downstream DNA are about 120° and 60°, respectively, in y-mtPIC (blue axes) and y-mtIC (red axes); i.e., the DNA is bent by ~60° in PIC and subsequently by another 60° to ~120° in the IC structure. The DNA bending calculations were done using CURVES+ server (11) . Movies S1-S3 shows the conformational changes during the transition from PIC to IC state. 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. 

Insights into transcription: structure and function of singlesubunit DNA-dependent RNA polymerases

Maintenance and Expression of Mammalian Mitochondrial DNA

Structural basis of mitochondrial transcription

Mechanism of bacterial transcription initiation: RNA polymerase -promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis

Mechanism of transcription initiation by the yeast mitochondrial RNA polymerase

Structural Basis of Mitochondrial Transcription Initiation

The thumb subdomain of yeast mitochondrial RNA polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding

Crystal structure of the transcription factor sc-mtTFB offers insights into mitochondrial transcription

Transcription factor-dependent DNA bending governs promoter recognition by the mitochondrial RNA polymerase

Mutations in the yeast mitochondrial RNA polymerase specificity factor, Mtf1, verify an essential role in promoter utilization

Structure of a transcribing T7 RNA polymerase initiation complex

Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism

Movies S1 -S3 Movie S1. Overall structural change in the transition from PIC to IC. Morphing between the transcription pre-initiation state (y-mtPIC, gray RPO41 and DNA, yellow MTF1) and initiation state (y-mtIC, yellow MTF1, blue RPO41, cyan non-template, and pink template) simulates the conformational changes in the promoter and protein during transition from the PIC to IC state. The downstream DNA bends inward and parts of the C-terminal domain including fingers (in front) undergo large conformational changes. MTF1, upstream DNA, and parts of the N-terminal domain of RPO41 that interact with MTF1 and upstream DNA show minimal conformational changes; e.g. the thumb helix on the left and MTF1 on the top have minimal movements.

Morphing between the PIC state (gray DNA) and IC state (cyan non-template, pink template, and 2-mer RNA pppGpG and UTPaS in stick models) shows DNA bubble expansion associated with the template base-pairing with the 2-mer RNA and UTP at the polymerase active site. The downstream DNA bends by about 60° (Fig. S5) . The protein atoms are removed for clear visualization of the DNA.Movie S3. Scrunching of the non-template DNA strand as an NT-loop. Morphing between the PIC state (gray DNA) and IC state (cyan non-template, pink template, and 2-mer RNA pppGpG and UTPaS in stick models) shows looping of the non-template strand into an NT-loop. This looping appears to be a major contributor to bending of the downstream DNA with respect to the upstream DNA (Fig. S5) .