{"paper_id":"23504aa9-0e8d-4175-b667-114eb29a697c","body_text":"Structural Basis of Mitochondrial Transcription Regulation via Interactions of \nPolRMT and TFAM with Upstream Promoter DNA \nAuthors and affiliations \nRory E. Shakey 1, Caitlin Schroeder 2, Xiangyu Deng 1, Jamie Smith 1, Alfredo J. \nHernandez2, Yang Gao1 \n1 Department of BioSciences at Rice University, Houston, TX 77005, USA \n2 Department of Biology, Tufts University, Medford, MA 02130, USA \n \nCorresponding author: Yang Gao, yg60@rice.edu \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nAbstract \nMitochondrial DNA (mtDNA) transcription is essential for cellular energy production and \nis carried out by a streamlined transcription system in which transcription factor A (TFAM), \ntranscription factor B2 (TFB2M), and the mitochondrial RNA polymerase ( PolRMT) \nassemble at defined promoters to initiate transcription. Previous structural studies \nelucidated the core initiation mechanism but relied on truncated promoter templates that \nexcluded upstream regulatory DNA interactions. Here, we present two conformati ons of \nmitochondrial transcription initiation complexes assembled on the heavy-strand promoter \n(HSP): a TFAM-bound complex with extended upstream DNA and a TFAM-free complex \ncontaining short linear DNA.  The TFAM -bound structure  reveals a transcription-\nstimulatory interface between PolRMT and the upstream promoter region (UPR) enabled \nby TFAM-induced promoter bending. Consistent with this structural observation , UPR \ntruncation reduces transcription from all mtDNA promoters, an effect abolished by \nmutation of the PolRMT interface. In contrast, the TFAM -free structure reveals a \ntranscription-inhibitory interaction of linear upstream DNA with the PolRMT tether helix, \nwhich would sterically clash with TFAM binding. Deletion of the tether helix increases off-\ntarget transcription, supporting an autoinhibitory role that enhances promoter specificity. \nTogether, these findings reveal how TFAM -shaped promoter architecture and PolRMT \nregulatory elements coordinate mitochondrial transcription initiation and regulation. \nGraphical Abstract \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nIntroduction \nMitochondria orchestrate cellular life and death signals through energy production, \nimmune signaling, and apoptotic pathways (1, 2). Central to these diverse functions is the \nexpression of the mitochondrial  genome (mtDNA), a compact and evolutionarily distinct \ngenome housed inside the organelle (3, 4). Transcription of mtDNA results in expression \nof 13 core proteins involved in oxidative phosphorylation (OXPHOS), the key energy -\nproducing pathway, as well as 22 tRNAs, 2 rRNAs, and mtDNA replication primers (3–5). \nAs such, dysregulation of mitochondrial transcription has been linked to various disorders, \nincluding neurodegenerative and cardiovascular diseases as well as cancer (6–9).  \n \nIn stark contrast to the multi-subunit, highly complex nuclear transcription machinery (10), \nmitochondrial transcription initiation relies on just three proteins (4, 11, 12). Transcription \nis carried out by PolRMT, a nuclear-encoded single-subunit RNA polymerase, structurally \nrelated to the T7 bacteriophage RNAP (13). Unlike the T7 RNAP, transcription initiation \nby PolRMT requires two transcription factors: transcription factor A (TFAM), which binds \nto and bends promoter DNA (14–16), and transcription factor B2 (TFB2M), which interacts \nwith PolRMT near the transcription start site (TSS) to stabilize the transcription bubble  \n(15, 17–19). Mitochondrial transcription is initiated at three promoter sequences, known \nas the heavy-strand promoter (HSP), the light-strand promoter (LSP), and the light-strand \npromoter 2 (LSP2)  (20). The HSP is the princi pal promoter for mitochondrial gene \nexpression as it encodes for 12 of the 13 mRNAs, 14 of the 22 tRNAs, and both of the \nrRNAs, while LSP and LSP2 encode the remain ing RNAs (3, 4, 11) . Additionally, \nregulation of transcription initiation is affected by TFAM-mediated compaction of mtDNA \ninto nucleoids, where tight nucleoid compaction inhibits transcription (21). Furthermore, \nthe non-coding 7S RNA was recently shown to stimulate formation of a transcriptionally-\ninactive PolRMT dimer (22). While there are far fewer components of mitochondrial \ntranscription initiation relative to nuclear transcription, the mechanism s of mitochondrial \ntranscription regulation are nuanced and require further elucidation.  \n \nDespite extensive biochemical and structural analyses of mitochondrial transcription \ninitiation complexes (mtTICs)  (15, 23 –26), critical aspects of promoter engagement  \nremain unresolved. Notably, previously solved structures (15, 23, 24) relied on promoter \ntemplates derived from the LSP and limited to the -40 to -50 position relative to the TSS, \npotentially excluding upstream regulatory DNA interactions. However, DNase-footprinting \nhas shown that mtTICs protect DNA further upstream (-50 to -60) (20, 27, 28). Consistent \nwith this  observation, in vitro  transcription using longer promoter DNA templates has \ndemonstrated enhanced  transcription activity from  both LSP and HSP , with  the effect \nbeing most pronounced for HSP (27). Moreover, the enhanced transcription for HSP was \nproposed to involve binding of an additional TFAM molecule and looping of the extended \nDNA (27). Together, these findings suggest that intrinsic elements of the upstream DNA, \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nparticularly for the HSP, and PolRMT contribute to regulation in ways not captured by \ncurrent structures.  \n \nCurrent structures have established interaction between TFAM and the PolRMT tether \nhelix (15, 23, 24), suggesting an essential role of TFAM in promoting PolRMT binding and \nmtTIC assembly. Paradoxically, although TFAM is considered essential for  canonical \nmtTIC formation, biochemical and structural studies have demonstrated that a stable and \nactive transcription complex can form in its absence (23, 25, 27, 29). Further, studies on \ntruncation of the PolRMT N-terminal extension (NTE), which contains the tether helix, \nhave shown enhanced transcription activity in Drosophila (30) and increased off-target \ntranscription initiation of mouse PolRMT in vitro, suggesting an additional regulatory role \nof the NTE (25). Elucidating the interactions between TFAM, PolRMT, and promoter DNA \nis required to clarify how these elements regulate transcription initiation and specificity. \n \nHere, we present cryo-EM analysis of mtTICs with an extended HSP template , which \nrevealed two distinct conformations  that identify  key regulatory elements . The first \nstructure, HSP with TFAM, resolves an interaction of PolRMT with extended upstream \npromoter DNA, which we have termed the upstream promoter region (UPR). Consistent \nwith the structural interface , templates containing the UPR enhanced transcription from \nall three promoters in vitro . Further, mutation  of three key lysine residues \n(K425E/K428E/K432E) at the PolRMT interface ablated UPR-mediated transcription \nenhancement. The second conformation showed a complex with short, linear upstream \nDNA engaged by the PolRMT tether helix  but lack ing TFAM. Tether helix truncation \nincreased nonspecific initiation, revealing an intrinsic autoinhibitory mechanism that \nenforces promoter specificity. Together, our study establishes the upstream DNA as an \nactive regulatory element  of mtDNA transcription  and reveal s how TFAM -dependent \npromoter architecture and intrinsic PolRMT elements cooperate to control mitochondrial \ntranscription initiation and specificity. \n \nMaterial and Methods \nCloning and Expression of TFAM, TFB2M, and PolRMT variants \nHuman TFAM (residues 43-246) with an N-terminal 6X-His tag and PreScission Protease \ncleavage sequence was inserted into the pET -28p vector via In-fusion cloning (Takara \nBio). Expression was performed in E. coli BL21(DE3) and cultures were grown in LB at \n37ºC until reaching optical density (OD600) of 0.6-0.8. Cultures were cooled on ice for 30 \nmin. Then, expression was induced with 1 mM IPTG , and cultures were incubated in  a \nshaker at 20ºC for 18 hours.  \n \nHuman TFB2M (residues 21-396) was inserted into the pET -SUMO vector with an N -\nterminal 6X-His-SUMO2 tag via In-fusion cloning. Expression was performed in E. coli \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nBL21(DE3) and cultures were grown in LB at 37 ºC to OD600 = 0.6-0.8. Cultures were \ncooled on ice for 30 min. Then, expression was induced with 0.5 mM IPTG, and cultures \nwere incubated in a shaker at 16ºC for 18 hours.  \n \nHuman PolRMT WT (residues 44-1230) was inserted into the pMAL-c6T (NEB) vector \nwith an N-terminal 6X-His-MBP tag and PreScission protease cleavage sequence via In-\nfusion cloning. The vector was transformed into ArcticExpress  (Agilent) cells containing \nthe pTF16 plasmid (Takara Bio) expressing TriggerFactor to enhance solubility. Cultures \nwere grown in LB containing 0.5 mg/mL L-Arabinose and 20 g/L glucose at 37ºC to OD600 \n= 0.3. Cultures were cooled on ice for ~2 hours before inducing expression with 0.1 mM \nIPTG, then shaking continued at 10ºC for ~40 hours. \n \nThe K425E/K428E/K432E (3KE)  PolRMT mutant construct was generated from the \nplasmid containing WT via In-fusion cloning. The tether helix truncation mutant (deletion \nof PolRMT residues 122-146 [ΔTH]) was generated via inverse PCR and the KLD enzyme \nmix (NEB) (primers included in Table S1). \n \nPurification of TFAM, TFB2M, and PolRMT variants \nTFAM was purified by first resuspending cell pellets in buffer N1 (20 mM Tris pH 7.5, 1 M \nNaCl, 1 mM BME, 10 mM Imidazole, and 10% glycerol) with 1 mM PMSF, 5 μg/mL DNase, \nand one Pierce EDTA-free protease inhibitor cocktail tablet. After lysis by sonication, the \nclarified lysate was incubated for 30 minutes with Ni 2+ resin (0.5 mL per 1 L cell culture). \nThe mixture was added to a gravity column, washed with buffer N2 (20 mM Tris pH 7.5, \n1 M NaCl, and 10% glycerol) containing 30 mM imidazole, and eluted with buffer B \ncontaining 300 mM imidazole. The eluent was incubated with PreScission protease at \n4ºC for 2 hours to cleave the His tag. The solution was diluted to 167 mM NaCl in MS \nbuffer (20 mM Tris pH 7.5 and 3 mM DTT) and loaded onto a Mono S 10/100 GL (Cytiva) \ncolumn equilibrated in MS buffer with 167 mM NaCl. TFAM was eluted with a gradient of \nMS buffer containing 1 M NaCl, with peak elution near 500 mM NaCl. Peak fractions were \nconcentrated, aliquoted, and stored at -80ºC. \n \nTFB2M was first purified by nickel affinity as described for TFAM. The eluent was then \nincubated with SenP2 at 4 ºC for 2 hours to cleave the His -SUMO tag. The sample was \ndiluted to 250 mM NaCl in Hep buffer (40 mM Tris pH 7.5 and 3 mM DTT) and loaded \nonto a 5 mL HiTrap Heparin HP (Cytiva) column equilibrated in Hep buffer containing 250 \nmM NaCl. TFB2M was eluted with a gradient of Hep buffer containing 1 M NaCl, with \npeak elution near 500 mM NaCl. Peak fractions were concentrated, aliquoted, and stored \nat -80ºC. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nPolRMT WT, 3KE, and ΔTH were purified by resuspending cells in buffer M1 (1X PBS, 1 \nM NaCl, 20% glycerol, 5 mM EDTA, 3 mM DTT, and 1 mM PMSF) containing 5 μg/mL \nDNase and 1 Pierce EDTA -free protease inhibitor cocktail tablet. Cells were lysed by \nsonication, and the clarified lysate was incubated for 1.5 hours with Amylose resin (250 \nμL per 1 L cell culture). The mixture was loaded onto a gravity column, washed with buffer \nM2 (40 mM Tris pH 8.0, 1 M NaCl, 20% glycerol, 1 mM EDTA, 5 mM DTT, and 1 mM \nPMSF), and eluted by incubating overnight with buffer M3 (40 mM Tris pH 8.0, 1 M NaCl, \n20% glycerol, 1 mM EDTA, 5 mM DTT, 1 mM PMSF, and 300 mM Maltose). The eluent \nwas diluted to 250 mM NaCl in QH buffer (40 mM Tris pH 8.0, 5 mM DTT, 1 mM EDTA, \nand 10% glycerol) and loaded onto two columns connected in series: 1 mL HiTrap Capto \nQ column (Cytiva) followed by 5 mL HiTrap Heparin HP column . The columns were \nequilibrated in QH buffer containing 250 mM NaCl. After loading, the Capto Q column, \nwhich contained contaminating nucleic acid, was removed prior to elution. Finally, the \nheparin column was eluted with a gradient of QH buffer with 1 M NaCl, with peak elution \nnear 400 mM NaCl. Peak fractions were concentrated, aliquoted, and stored at -80ºC. \n \nIn Vitro Transcription Initiation Assay \nDNA templates for in vitro  transcription assays were generated by annealing \ncomplementary oligonucleotides (IDT) (Table S1 ). Transcription  initiation reactions \ncontained 20 nM DNA , 100 nM TFB2M, 60 nM TFAM, and 0-300 nM PolRMT variant \n(presence of TFAM and PolRMT concentrations are indicated in figures) in transcription \nbuffer (40 mM Tris pH 8.0, 10 mM MgCl2, 100 μg/mL BSA, 1 mM DTT, and 2 U/μL RNase \ninhibitor, Murine [NEB]) with 500 μM ATP/CTP/GTP, 10 μM cold UTP, and 33.3 nM 32P a-\nUTP. Reactions were incubate d for 30 min at 30ºC and stopped by the addition of 2X \nUrea-PAGE loading buffer (90% formamide, 50 mM EDTA, 0.1% xylene cyanol, and 0.1% \nbromophenol blue) followed by incubation at 95ºC for 10 min. Reactions were resolved \nby UREA -PAGE on a 20% polyacrylamide gel in 1X TBE buffer with 30 min pre -run \nfollowed by a  1.5-hour run at 250V.  Gels were exposed to phosphor imaging screen \novernight, scanned, and quantified using a Sapphire Biomolecular Imager and Azure Spot \n(Azure Biosystems). \n \nPromoter-Free In Vitro Transcription Elongation Assay \nElongation scaffolds for in vitro  transcription elongation assays were generated  by \nannealing DNA oligonucleotide pairs with a 5' -FAM-labelled RNA oligonucleotide (IDT)  \n(Table S1). Annealing was performed by mixing the non-template strand, template strand, \nand RNA in a 2:1.5:1 molar ratio, heating to 90ºC for 5 min, cooling to 60ºC at a rate of -\n0.5ºC per min, holding at 60ºC for 20 min, followed by cooling to 12ºC at -0.5ºC per min. \nTranscription elongation reactions contained 20 nM annealed elongation scaffold and 0-\n250 nM PolRMT variant (concentrations are indicated in figures) in transcription buffer \nwith 500 μM ATP/CTP/GTP/UTP. Reactions were incubated for 20 min at 30 ºC and \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nstopped by the addition of 2X Urea -PAGE loading buffer followed by incubation at 95 ºC \nfor 10 min. Reactions were resolved by UREA -PAGE on a 22.5% polyacrylamide gel in \n1X TBE buffer with 30 min pre-run followed by a 1.5-hour run at 250V. Gels were scanned \nand quantified using a Sapphire Biomolecular Imager and Azure Spot. \n \nDouble Filter Binding Assay \nThe 7S RNA substrate for binding assays was in vitro  transcribed using T7 RNAP , \ndephosphorylated with calf intestinal phosphatase (NEB), 5' end-labeled with [γ-32P] ATP \n(Revvity) using T4 polynucleotide kinase (NEB), and purified by denaturing PAGE. 0.1 \nnM radiolabeled 7S RNA was incubated with PolRMT (0-300 nM) in binding buffer (40 \nmM HEPES pH 7.5, 100 mM KCl, 20 mM β-ME) at 25ºC for 15 minutes. Reactions were \nvacuum-filtered through nitrocellulose (0.45 μm, Schleicher & Schuell) and nylon \n(Whatman) membranes in a dot-blot microfiltration apparatus (Bio-Rad) (31). Membranes \nwere air-dried and quantified by phosphorimaging (Cytiva). The fraction of RNA bound \nwas calculated by the ratio of counts on nitrocellulose to total counts (nitrocellulose + \nnylon) (32).  \n \nCryo-EM Sample Preparation \nThe complex was assembled by mixing 3.3 μM TFAM, 5 μM TFB2M, 3.3 μM PolRMT WT, \nand 3.6 μM HSP -60 to +11 DNA substrate yielding a final concentration for the complex \nof 1 mg/mL in buffer containing 40 mM Tris (pH 8.0), 10 mM MgCl2, 10 mM DTT, and 100 \nmM NaCl. After 30 min incubation on ice and centrifugation at 17,000 x g for 10 min, a 3 \nμL drop of the complex was spotted onto freshly glow -discharged grids (Quantifoil R \n1.2/1.3 Cu 300 mesh grids). Excess sample was blotted using the Vitrobot Mark IV (FEI) \nwith the standard Vitrobot filter paper (Ø55/20 mm [Ted Pella]), with blotting time 2 s and \nblotting force 3 under 100% humidity at 20ºC. The grids were flash-frozen in liquid ethane \nand stored in liquid nitrogen. \n \nCryo-EM Data Collection and Processing \nThe dataset of 27,688 movies was collected from Stan ford-SLAC Cryo-EM Center and \nrecorded on a Titan Krios G4i electron microscope operated at 300 kV with serial EM (33) \n(detailed parameters for data collection are summarized in Table 1). Motion correction \nwas performed with MotionCor2 (34), and defocus values were estimated on non -dose-\nweighted micrographs with Gctf (35). For processing of the dataset, reference-free auto-\npicking (Laplacian -of-Gaussian picking)  was performed in RELION -4.0 (36, 37) . \n11,635,618 particles were picked and extracted to pixel size 4.79 Å/pixel and imported to \ncryoSPARC-4.2 for 2D classification  for multiple rounds of 2D classification and subset \nselection. 7,872,349 particles were used for ab-initio reconstruction and separated into \nsix classes. Four of those classes were subjected to heterogeneous refinement, where \none class showed clear features for PolRMT, TFB2M, and DNA. Particles from this class \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nwere further cleaned with 2D classification and split into two classes by ab-initio \nreconstruction and heterogeneous refinement. The first class was further processed to \nyield the HSP without TFAM complex, while the second class was used for the HSP with \nTFAM complex. Particles from the first class with only density for short upstream DNA \nwere extracted (0.958 Å/pixel) in RELION-4.0 and reimported to cryoSPARC-4.2 for ab-\ninitio reconstruction into six classes followed by  heterogeneous refinement  and non-\nuniform refinement. Particles from the three classes with density for DNA were subjected \nto heterogeneous refinement with the best map (3.37 Å resolution) and worst map from \nthe previous non-uniform refinement. This yielded 446,280 particles, which were used for \nCTF refinement and non-uniform refinement resulting in a map with  a resolution of 2.86 \nÅ. Particles from the class with density for longer DNA  were extracted (0.958 Å/pixel) in \nRELION-4.0 and reimported to cryoSPARC-4.2 for ab-initio reconstruction into six classes \nfollowed by heterogeneous refinement and non-uniform refinement. One class yielded a \n3.51 Å resolution map with density for the extended DNA, which was used as a template \nfor Topaz (38) training and particle picking . In parallel, this class was subjected to local \n3D classification using a focused mask for the extended DNA, which resulted in  2 of 10 \nclasses with density for the DNA in contact with PolRMT. Non-uniform refinement of these \ntwo classes produced a 4.13 Å resolution map. Topaz particle picking resulted in \n32,940,939 particles which were extracted (0.958 Å/pixel) in RELION-4.0 and reimported \nto cryoSPARC-4.2 for 2D cleaning resulting in 23,147,478 particles. The particles were \nthen subjected to heterogeneous refinement with t he 4.13 Å resolution map combined \nwith five poor maps from previous ab-initio reconstruction. This produced one class with \nfeatures of PolRMT, TFB2M, TFAM and extended DNA, which was used for three rounds \nof iterative ab-initio reconstruction and heterogeneous refinement. This yielded 200,388 \nparticles, which were used for CTF refinement and non-uniform refinement resulting in a \nmap with a resolution of 3.33 Å. Both maps were subject to 3DFlex jobs (39) to improve \nlocal resolution and Phenix auto sharpen (40) to aid in model building. \n \nModel Building and Refinement \nFor the HSP with TFAM complex, the previously published structure of the TIC HSP \ncrystal structure (PDB: 6ERQ) was used as an initial model for manually docking into the \ncryo-EM density map using U CSF Chimera  (41). Additionally, an AlphaFold 3 (42) \nprediction of the mtTIC with the extended HSP substrate was used for modelling the DNA, \nand the recently published structures of the pre-IC3 (PDB 9GZM) and IC0 (9MN5) on LSP \nwere used for mode ling regions of the PolRMT NTE, thumb, and specificity loop and \nTFB2M NTD. The model was further manually rebuilt in COOT  (43) based on electron \ndensity and refined in Phenix  (44) with real-space refinement and secondary structure \nand geometry restraints. For the HSP without TFAM complex, the model of the HSP with \nTFAM complex was used as an initial model for manually docking into the cryo-EM density \nmap using Chimera. An AlphaFold 3 prediction of the mtTIC wi thout TFAM was used to \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nmodel the linear upstream DNA. As before, the model was manually rebuilt in COOT and \nrefined in Phenix. \n \nStatistical Analysis \nAll data were graphed and analyzed for significance using GraphPad Prism 10. For \ntranscription initiation reactions with PolRMT titration in Figure 2-3, background was \nsubtracted from intensity values and normalization was performed by dividing each \nintensity by the maximum intensity value from each individual experiment gel.  \nSignificance was determined using a 2-way ANOVA test with Šídák’s multiple \ncomparisons test. For transcription initiation reactions with a single concentration of \nPolRMT in Figure 4 and Figure S5, background was subtracted from raw values and \nnormalization was performed by dividing each intensity by the intensity value of the \nsample containing TFAM and WT PolRMT from each individual experiment gel. \nSignificance was determined using a 1-way ANOVA test with Tukey’s multiple \ncomparisons test. For transcription elongation reactions, elongation percentage was \ncalculated by dividing the intensity of the elongation product bands by the total intensity \nof the lane after subtracting background. Mean and SEM values were determined from \n3 independent replicates for all transcription reactions. For filter binding assays, binding \nmeasurements were fit to a one-site specific binding model to determine KD values \nbased on 5 independent replicates. To compare the KD values an extra sum-of-squares \nF test was performed and the null hypothesis was rejected for p-value < 0.05. \n \nProtein Sequence Alignments \nSequence alignments of PolRMT homologs and TFAM homologs for Figure S6 were \nperformed with constraint-based multiple alignment tool (COBALT) (45). \n \nResults \nCryo-EM structure of the Mitochondrial Transcription Initiation Complex on an \nextended Heavy-Strand Promoter \nAlthough extended promoter regions have been shown to stimulate mitochondrial \ntranscription initiation  (27), the underlying mechanism remains unclear . P revious \nstructural studies employed DNA substrates containing minimal promoter sequences  40 \nto 50-bp upstream of the TSS (15, 23, 24). To investigate the role of extended promoter \nDNA in mitochondrial transcription initiation, we performed single -particle cryo-EM \nanalysis of the mtTIC with an extended HSP template. The DNA substrate consisted of \nthe HSP sequence from -60 to +11, with a 7-bp bubble (-4 to +3) generated by mutating \nthe non-template strand (NT) , similar to the mtTIC crystal structure (15) (Fig. 1A). The \nHSP with TFAM mtTIC structure was determined at 3.33 Å resolution (Fig. S1 and Table \n1). We were able to resolve TFAM, TFB2M, and PolRMT along with DNA from the -57 to \n+11 position (Fig. 1B), including an extended linear DNA that approaches the N-terminal \ndomain (NTD) of PolRMT (Fig. 1C).  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nFigure 1. Cryo-EM structure of the Mitochondrial Transcription Initiation Complex on HSP \nwith extended upstream DNA.  (A) Schematic of the HSP bubble DNA substrate, TFAM, \nTFB2M, and PolRMT. The DNA substrate non-template strand (NT) and template strand \n(TS) are shown in cyan and slate, respectively, and shaded circles represent the resolved \nbases, bases that are not visible are represented by hollow circles. PolRMT is shown in \npink and divided into its domains: mitochondrial targeting sequence (MTS) (not included \nin expression constructs), N -terminal extension (NTE), tether helix in purple, \npentatricopeptide repeat domain (PPR), N-terminal domain (NTD), residues K425, K428, \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nand K432 in red, AT-rich recognition loop (AT-RRL), TFB2M-interacting hairpin (TFB2M \nhairpin), palm, thumb, and specificity loop. TFAM is shown in orange with the MTS, high \nmobility group box A (HMG -A), linker, HMG -B, and C -terminal tail (C -tail). TFB2M is  \nshown in green with the MTS, N-terminal domain, and C-terminal domain (CTD). (B) Left: \ncryo-EM density map of the mtTIC on the HSP substrate showing the extended upstream \nDNA with proteins and DNA colored as in (A). Right: 90 º rotation around the X -axis \nshowing the PolRMT active site and downstream DNA. (C)  Cartoon representation of \nDNA and proteins shown in (B). \n \nTable 1. Cryo-EM data collection, model refinement, and validation \n \nHSP TIC with \nTFAM \nHSP TIC without \nTFAM \nData collection and processing \nEMDB ID 75933 75934 \nPDB ID 11PR 11PS \nMicroscope  Titan Krios G4i (Δ) Titan Krios G4i (Δ) \nDetector  Falcon 4i Falcon 4i \nMagnification 146,000 146,000 \nVoltage (kV) 300 300 \nElectron exposure (e-/Å2) 50 50 \nDefocus range (µm) -0.8 to -2.0 -0.8 to -2.0 \nPixel size (Å) 0.958 0.958 \nNumber of movies 27,688 27,688 \nInitial particles (no.) 11,635,618 32,940,939 \nFinal particles (no.) 200,388 446,280 \nMap resolution (Å) 3.33 2.86 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nFSC (Fourier shell correlation) threshold 0.143 0.143 \nRefinement \nNo. of atoms 15733 12320 \nNo. of residues - - \nProtein 1606 1358 \nNucleic acid 136 70 \nRMSDs - - \nBond lengths (Å) 0.007 0.007 \nBond angles (º) 1.047 1.143 \nValidation \nMolProbity score 1.29 1.39 \nClashscore 5.43 6.99 \nRotamer outlier (%) 0.00 0.00 \nRamachandran plot (%) - - \nFavored 97.99 98.00 \nAllowed 2.01 2.00 \nOutliers 0.00 0.00 \n \n \nThe PolRMT and TFB2M core  in our transcription initiation complex adopt s a similar \nconformation as observed in previous structures (15, 23, 24). Alignment of PolRMT and \nTFB2M Cα atoms results in an RMSD of 1.1 Å over 1106 residues to the HSP crystal \nstructure (PDB: 6ERQ) and 0.9 Å over 893 residues to an LSP structure solved by cryo-\nEM (PDB: 9MN5) (Table S2) (15, 23, 24). The PolRMT fingers domain adopts a clenched \nconformation in our structure, which is consistent with other structures that do not have \nan incoming NTP bound (15, 24) (Fig. S2A). However, TFAM is rotated by 16º in our \nstructure compared to the HSP crystal structure (PDB: 6ERQ) and 12º to the LSP cryo-\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nEM structure (PDB: 9MN5), suggesting flexibility of TFAM within the mtTIC (15, 23, 24) \n(Fig. S2B and Table S2).  \n \nPolRMT interacts with the upstream promoter region during transcription initiation  \nIn our mtTIC structure, clear density can be observed for the HSP UPR, which forms an \nadditional interaction interface with PolRMT. Specifically, K425, K428, and K432, which \nare located in a helix of the PolRMT NTD, contact the DNA phosphate backbone through \nelectrostatic interactions (Fig. 2A). While our structure reveals this interaction on the HSP, \nDNase footprinting experiments have suggested that the interaction occurs on all three \nmitochondrial promoters (20). To test the role of the UPR in transcription initiation , we \ndesigned long DNA templates including the UPR to the -70 position and truncated \ntemplates that ended at the -40 position from all three promoters (Fig. 2B). An in vitro \ntranscription assay using the reconstituted mtTIC with PolRMT titration was employed to \nmeasure transcript production  from each DNA substrate. On  the LSP, the truncated \ntemplate had ~50% lower transcript produc tion (Fig. 2C). Similarly, template truncation \nled to a decrease in transcript production for both HSP and LSP2, with a 20% and 50% \nreduction, respectively (Fig. 2D-E). Together, these results show that the PolRMT NTD \ninteracts with the UPR and that this interaction is important for transcription initiation at \nall three mitochondrial promoters. \n \n \nFigure 2. PolRMT interacts with the upstream promoter region (UPR) during transcription \ninitiation. (A) Cryo -EM density map highlighting the interaction of PolRMT K425, K428, \nand K432 with the UPR, specifically, the DNA backbone of the TS at bases -50 and -51. \n(B) DNA substrate design for transcription initiation assays with the short template \nextending to the -40-bp and long template to the -70-bp. Representative gel image of \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nPolRMT titration from 0 -200 nM with the -40 and -70-bp LSP DNA template. (C) \nQuantification of transcript production from the LSP with -40 and -70-bp template. (D) \nQuantification of transcript production from the HSP with -40 and -70-bp template. (E) \nQuantification of transcript production from the LSP2 with -40 and -70-bp template. Mean \nand SEM were determined from 3 independent replicates.  Significance was determined \nusing a 2-way ANOVA test with Šídák’s multiple comparisons test. Significance: * p<0.05, \n** p<0.01, *** p<0.001, **** p<0.0001. \n \nMutation of PolRMT residues K425, K428, and K432 abolishes UPR enhancement \nof transcript yield \nTo directly test the role of PolRMT residues K425, K428, and K432 in mediating interaction \nwith the UPR, we mutated all three lysines to glutamate (K425E/K428E/K432E; 3KE) to \ndisrupt electrostatic interactions with the negatively charged DNA backbone . We then \nevaluated transcription initiation by the 3KE mutant using both long and truncated DNA \ntemplates. In contrast to WT PolRMT, transcript yield with the 3KE mutant was largely \ninsensitive to promoter truncation (Fig. 3A-C). For LSP, the 3KE mutant produced slightly \ngreater transcript yield from the shorter DNA template, opposite to WT PolRMT (Fig. 3A). \nOn both HSP and LSP2, 3KE PolRMT produced similar transcript yield  from both \ntemplates across all PolRMT concentrations tested (Fig. 3B and 3C). These results \nconfirmed that the interaction between PolRMT and the UPR is mediated primarily by the \ninteraction of PolRMT K425, K428, and K432 with the DNA backbone. \n \nTo test whether this interaction may also facilitate transcription elongation , we employed \na promoter -independent transcription elongation assa y. The elongation scaffold was \ndesigned with a 9-nt bubble between the NT and TS where the labeled RNA was paired \nwith the TS, as in previous studies (46, 47), and an upstream DNA length of either 40 or \n72-bp (Fig. 3D). The length of upstream DNA had no significant impact on transcription \nelongation for both WT and  the 3KE mutant ( Fig. 3 E and 3F). Taken together, this \nsuggests that the interaction of PolRMT with extended upstream DNA occurs only during \ntranscription initiation, likely facilitated by TFAM bending of promoter DNA into a U-shape. \nPrevious studies suggested that PolRMT binding to 7S RNA negatively regulates  \ntranscription initiation  (48). The structure of a transcription ally-inactive PolRMT dimer \nbound to 7S RNA suggested that, along with several other positively charged residues, \nK425, K428, and K432 may contact 7S RNA (48). To probe whether the positively charged \nresidues K425, K428, and K432 also contribute to transcription regulation via RNA \ninteractions, we evaluated binding of PolRMT to 7S RNA using a filter binding assay. We \nsaw a 3-fold decrease in binding affinity in the 3KE mutant compared to WT ( Fig. 3G). \nThese findings indicate that K425, K428, and K432  contribute not only to transcription \ninitiation but also to regulation via the extensive PolRMT–7S RNA binding interface. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\n \nFigure 3. Mutation of PolRMT K425/K428/K432 abolishes the enhancement of transcript \nyield by the UPR. (A) Quantification of PolRMT K425E/K428E/K432E (3KE) transcript \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nproduction from the LSP with -40- and -70-bp template. (B) Quantification of PolRMT 3KE \ntranscript production from the HSP with -40- and -70-bp template. (C) Quantification of \nPolRMT 3KE transcript production from the LSP2 with -40- and -70-bp template. Mean \nand SEM were determined from 3 independent replicates.  Significance was determined \nusing a 2-way ANOVA test with Šídák’s multiple comparisons test. Significance: ** p<0.01. \n(D) Elongation substrate used for promoter-free transcription elongation assays. The 12-\nnt FAM-labeled RNA is shown in red and the length of upstream DNA is indicated as either \n42-nt or 72 -nt. Representative gel of promoter -free transcription elongation assay with \ntitration of PolRMT from 0 -250 nM. The unelongated 12 -nt substrate and elongation \nproducts greater than 12-nt are indicated. (E) Quantification of promoter-free transcription \nelongation by WT PolRMT on the -72- and -42-nt elongation scaffolds. (F) Quantification \nof promoter -free transcription elongation by 3KE PolRMT on the -72- and -42-nt \nelongation scaffolds. Mean and SEM were determined from 3 independent replicates. (G) \nQuantification of filter binding assays for PolRMT WT and 3KE binding to 7S RNA. Mean \nand SD were determined from 5 independent replicates.  \n \nCryo-EM Structure of the Mitochondrial Transcription Initiation Complex without \nTFAM \nDuring cryo-EM data processing (Fig. S1 and Table 1), we observed a second class of \nmtTIC in which TFAM was not present and short linear upstream DNA was observed (Fig. \n4A and Fig. S3). This structure was resolved to 2.86 Å resolution, and it lacked density \nfor DNA upstream of position -24 and the PolRMT N-terminal extension (NTE, residue s \n44-121 and 147 -217) (Fig. 4A). Notably, we observed electron density adjacent to the \nshort, linear upstream DNA that can accommodate the PolRMT tether helix, which \ncontains several positively charged re sidues and could interact with the DNA backbone  \n(Fig. S4). Aside from the extra density and slightly longer upstream DNA, the core \nPolRMT and TFB2M of this structure were nearly identical to our structure containing \nTFAM with an RMSD of 0.6 Å for the 1331 Cα atoms (Table S2 and S3).  \n \nThe tether helix of PolRMT has an autoinhibitory role in transcription initiation and \ncontributes to specificity \nThe apparent density for the PolRMT tether helix near the linear upstream DNA suggested \na possible role in transcription initiation in the absence of TFAM. To test this, we generated \na PolRMT mutant lacking the tether helix (residue s 122-146) (ΔTH). Previous work on \nmouse PolRMT demonstrated that truncation of the entire N-terminal extension (including \nthe tether helix) led to off -target transcription initiation  (25). To test nonspecific \ntranscription (NS) initiation, we employed a DNA fragment from the mtDNA genome that \ncontains the conserved AAAGA sequence present  at positions +1 to +5 on all three \npromoters and occurs at over 30 locations throughout the mtDNA genome (Fig. 4B). We \nperformed in vitro  transcription assay s to test the transcript yield  from WT and ΔTH \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nPolRMT in both the presence and absence of TFAM. The ΔTH mutant had significantly \nhigher NS transcript  yield than WT, independent of the presence of  TFAM (Fig. 4 C). \nInclusion of TFAM significantly decreased NS transcription of WT PolRMT, while only a \nslight but not significant reduction was observed for ΔTH (Fig. 4C). Further, we used this \nassay to test each of the three mitochondrial promoters. On LSP, inclusion of TFAM \nincreased transcript yield by  WT PolRMT, while ΔTH had similar yield to WT in the \nabsence of TFAM and was not significantly enhanced by the presence of TFAM (Fig. 4D). \nOn HSP, ΔTH had significantly greater TFAM-independent transcription yield than WT, \nwhile TFAM-dependent transcription  yield was slightly higher than WT  (Fig. 4 E). On \nLSP2, ΔTH had greater transcript production in both the presence and absence of TFAM \ncompared to WT ( Fig. 4 F). A similar trend was also observed when the PolRMT \nconcentration was increased  (Fig. S5 ). Additionally, promoter -free transcription \nelongation was nearly identical for WT and ΔTH PolRMT (Fig. 4G). Taken together, these \nresults suggest that  the PolRMT tether helix has an autoinhibitory role in transcription \ninitiation from the HSP and LSP2 promoters, and that it contributes to  the specificity of \ntranscription initiation by limiting nonspecific initiation.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\n \nFigure 4. The PolRMT tether helix has an autoinhibitory effect on nonspecific transcription \ninitiation and initiation from the HSP and LSP2. (A) Left: cryo-EM density map of the \nmtTIC without TFAM, and a short linear upstream DNA with adjacent electron density that \naccommodates the PolRMT tether helix. Right: Cartoon representation of DNA and \nproteins colored as in ( Fig. 1A). (B) DNA sequence for nonspecific (NS) transcription \ninitiation template that contains the +1 to +5 sequence conserved amongst all three \npromoters (highlighted yellow) and is derived from  the mtDNA genome, positions 4211-\n4300 (3). Representative gel image of transcription initiation by PolRMT WT or ΔTH in \nthe presence or absence of TFAM on the NS DNA template with the 20 -nt transcription \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nproduct indicated. Transcription reactions contained 20 nM DNA template, 100 nM \nTFB2M, 0 or 60 nM TFAM, and 0 or 100 nM PolRMT. (C) Quantification of transcript  \nproduction from the NS DNA substrate. ( D) Quantification of transcript production from \nthe LSP. (E) Quantification of  transcript intensity  from the HSP. (F) Quantification of \ntranscript production  from the LSP2. ( G) Quantification of promoter -free transcription \nelongation by WT and ΔTH PolRMT on a -42-nt elongation scaffold. Mean and SEM were \ndetermined from 3 independent replicates. Significance was determined using a 1 -way \nANOVA test with Tukey’s multiple comparisons test. Significance: * p<0.05, ** p<0.01, **** \np<0.0001. \n \nDiscussion \nHuman mtDNA transcription  initiation is a streamlined, three -component system that \nrelies on the coordinated activities of PolRMT, TFB2M, and TFAM. TFAM is unique among \ntranscription factors in that it  also functions  as the principal mitochondrial genome -\npackaging protein (49). Here, we demonstrate that the TFAM-induced U-shaped DNA \narchitecture enables additional protein–DNA contacts with PolRMT at the UPR, promoting \nproductive assembly of the mtTIC. Consistent with this, the UPR enhances transcription \ninitiation on all three mitochondrial promoters, and this stimulatory effect is abolished by \nmutations of the PolRMT interface that contacts the UPR. Importantly, this interaction is \nspecific to initiation, as elongation proceeds independently of TFAM and is unaffected by \nthese mutations. Notably, the same PolRMT interface also contributes to regulation by 7S \nRNA (48), as mutations reduce 7S RNA binding, suggesting a shared regulatory surface. \n \nThe HSP and LSP in the mitochondrial genome are separated by a 153 -bp non-coding \nregion. Previous footprinting studies suggested that multiple TFAM molecules may bind \nwithin this region to stimulate transcription from both promoters  (27). However, in \navailable TIC structures  (15, 23, 24) , TFAM-induced U-shaped DNA bending positions \nthe immediate upstream DNA in close proximity to the PolRMT surface, such that \nadditional TFAM binding at positions −30 to −60 would sterically clash. Instead, the ~30-\nbp region between the two  UPRs could accommodate additional TFAM occupancy . \nAdditional TFAM binding at these distal sites may further remodel the non-coding region, \npotentially bringing the LSP and HSP initiation complexes into spatial proximity (Fig. 5A). \nThis could generate a transcriptional hub that coordinates promoter usage and enable s \nregulatory crosstalk, similarly to transcription by yeast RNA Pol II, which forms a dimer of \nthe polymerase -mediator complexes simultaneously on two divergent promoters  (50). \nWhile PolRMT dimerization via 7S RNA inhibits transcription (48), a different inter face \nwas found for dimerization of the apo protein (23), and it is unknown if a higher-order \norganization of both promoters may involve transcriptionally productive PolRMT \ndimerization. Defining this higher -order topology will require future structural and \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nbiochemical studies aimed at visualizing multi-TFAM assemblies and both LSP and HSP \nTICs on extended promoter templates. \n \nAnother aspect of transcription initiation regulation is the engagement of TFAM and the \nPolRMT N-terminal tether helix. In the absence of TFAM, we observe the highly positively \ncharged tether helix positioned near the upstream DNA, which remains in a linear \nconformation. However, this positioning would sterically clash with TFAM -induced U -\nshaped DNA bending, indicating that the tether helix –DNA interaction is mutually \nexclusive with pro ductive TFAM binding. Functional assays show that deletion of the \ntether helix increases nonspecific transcription as well as both TFAM -dependent and \nTFAM-independent initiation from the HSP and LSP2, supporting an autoinhibitory role. \nThe nonspecific DNA engagement of the tether helix may inhibit PolRMT activity through \nimpaired ability to scan for the specific TSS. This is analogous to observations of p53 and \nother DNA-binding proteins that have both specific and nonspecific DNA interactions (51, \n52). Alternatively, tether helix–DNA binding may disfavor conformations required for TSS \nmelting (19, 53, 54) . Furthermore, the tether helix may adopt intramolecular contacts \nwithin PolRMT that reduce nonspecific DNA binding. Upon TFAM binding, the tether helix \nis displaced from DNA through electrostatic interaction with TFAM’s acidic surface  (Fig. \nS4), thereby relieving inhibition. Although the precise mechanism remains to be defined, \nour data support a model in which the tether helix suppresses spurious initiation and is \nrepositioned only when proper promoter architecture with TFAM binding is established. \n \nBecause TFAM binds DNA with limited sequence specificity and is highly abundant in \nmitochondrial nucleoids, a key question is how nonspecific transcription is avoided. Our \nfindings suggest a multilayered mechanism. First, the tether helix suppresses basal \nPolRMT transcription on nonspecific templates  (Fig. 5B). Second, productive initiation \nrequires a precise DNA geometry generated by TFAM -induced U -bending and UPR \ncontact with PolRMT. It has been shown that TFAM also contributes to this specificity as \nthe mitochondrial promoters allow for greater DNA bending and complex stability than \nnonspecific DNA (55, 56). Additionally, high TFAM occupancy of mtDNA directly inhibits \nPolRMT access and transcription (21). Together, these features establish a coincidence-\ndetection mechanism in which DNA sequence, TFAM -mediated architecture, and \npolymerase conformational control must align to permit specific and efficient initiation. \n \nEvolutionarily, TFAM was not originally a dedicated transcription factor. The yeast \nhomolog, Abf2p, primarily functions in mitochondrial genome packaging  (57–59), and \nmitochondrial RNA polymerases in these species lack a comparable N -terminal tether \nhelix (Fig. S6). This correlation suggests that acquisition of the tether helix and the \ntranscriptional role of TFAM co-evolved to enable tighter regulatory control in metazoan \nmitochondria. As mitochondrial genomes became more compact and gene expression \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nmore tightly coordinated with cellular physiology, TFAM-mediated genome packaging and \ntranscription became functionally integrated, with TFAM coupled to an autoinhibitory \npolymerase element. This arrangement may have provided an efficient means to enhance \npromoter specificity within a minimal transcription system. \n \nIn summary, our study demonstrates that extended upstream promoter DNA directly \nengages PolRMT to enhance transcription initiation, while the tether helix functions as an \nautoinhibitory module that enforces promoter specificity. By integrating structural and \nbiochemical analyses, we provide a refined model in which TFAM -mediated DNA \narchitecture an d PolRMT regulatory elements cooperate to control initiation. More \nbroadly, these findings illustrate how modular extensions appended to a bacteriophage -\nlike RNA polymerase can introduce regulatory complexity into a streamlined transcription \napparatus. Future studies aimed at defining higher-order promoter DNA organization and \nregulatory RNA interactions will further clarify how mitochondrial transcription is \ndynamically tuned under physiological and pathological states. \n \nFigure 5. Model for regulation of mtDNA transcription. (A) Assembled mtTICs at both the \nLSP and HSP ends of the inter-promoter region, with dashed lines to indicate the potential \nfor additional TFAM binding and topological rearrangement. ( B) (Left) PolRMT and \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nTFB2M alone can assemble a mtTIC where the PolRMT tether helix interacts with linear \nupstream DNA resulting in low activity, likely due to impaired search for the proper TSS,  \nand moderate specificity. (Middle) Truncation of the PolRMT tether helix allows for high \ntranscription activity, possibly due to a  faster search for a TSS sequence , but the \nspecificity is poor. (Right) The binding of TFAM to promoter DNA and interaction with the \nPolRMT tether helix contributes to highly specific transcription initiatio n while also \nincreasing activity through PolRMT interaction with the UPR. \n \nData Availability \nAll original data and materials are available upon request. The atomic coordinates for the \nHSP with TFAM complex and HSP without TFAM complex have been deposited to the \nProtein Data Bank under accession code PDB: 11PR and PDB: 11PS, respectively. The \ncorresponding cryo-EM density maps have been deposited to the Electron Microscopy \nData Bank under accession code EMDB: 75933 and EMDB: 75934, respectively.  \nAcknowledgements \nWe thank Dr. Gaya P. Yadav at the Laboratory for Biomolecular Structure and Dynamics \n(LBSD) of Texas A&M University and the core facility at Stanford-SLAC Cryo-EM Center \nfor cryoEM data collection. \nFunding \nThe work was supported by the National Institutes of Health [35GM142722 to Y.G.], and \nTufts Faculty Startup Research Fund to AJH. \nAuthor contributions RES purified recombinant proteins, performed in vitro transcription \nassays, cryo-EM sample preparation, data processing, model building, refinement, and \nstatistical analysis, wrote and edited manuscript . CS generated the 7S RNA substrate \nand performed filter binding assays. XD performed preliminary cryo-EM data processing. \nJS performed cloning for TFAM and TFB2M. AJH reviewed and edited manuscript. YG \nconceptualized and administered project, wrote and edited manuscript.  \nConflict of interest statement \nNone declared. \nReferences \n1. West,A.P ., Khoury-Hanold,W., Staron,M., Tal,M.C., Pineda,C.M., Lang,S.M., Bestwick,M., \nDuguay,B.A., Raimundo,N., MacDuF,D.A., et al. (2015) Mitochondrial DNA stress \nprimes the antiviral innate immune response. Nature, 520, 553–557. \n2. Ott,M., Gogvadze,V ., Orrenius,S. and Zhivotovsky,B. (2007) Mitochondria, oxidative stress \nand cell death. Apoptosis, 12, 913–922. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\n3. Anderson,S., Bankier,A.T., Barrell,B.G., de Bruijn,M.H.L., Coulson,A.R., Drouin,J., \nEperon,I.C., Nierlich,D.P ., Roe,B.A., Sanger,F ., et al. (1981) Sequence and \norganization of the human mitochondrial genome. Nature, 290, 457–465. \n4. Taanman,J.-W. (1999) The mitochondrial genome: structure, transcription, translation \nand replication. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1410, 103–\n123. \n5. Wanrooij,S., Fusté,J.M., Farge,G., Shi, Y ., Gustafsson,C.M. and Falkenberg,M. (2008) \nHuman mitochondrial RNA polymerase primes lagging-strand DNA synthesis in \nvitro. Proc Natl Acad Sci U S A, 105, 11122–11127. \n6. Van Haute,L., O’Connor,E., Díaz-Maldonado,H., Munro,B., Polavarapu,K., Hock,D.H., \nArunachal,G., Athanasiou-Fragkouli,A., Bardhan,M., Barth,M., et al. (2023) TEFM \nvariants impair mitochondrial transcription causing childhood-onset neurological \ndisease. Nat Commun, 14, 1009. \n7. Lei,T., Rui, Y ., Xiaoshuang,Z., Jinglan,Z. and Jihong,Z. (2024) Mitochondria transcription \nand cancer. Cell Death Discov., 10, 168. \n8. Oláhová,M., Peter,B., Szilagyi,Z., Diaz-Maldonado,H., Singh,M., Sommerville,E.W., \nBlakely,E.L., Collier,J.J., Hoberg,E., Stránecký,V ., et al. (2021) POLRMT mutations \nimpair mitochondrial transcription causing neurological disease. Nat Commun, 12, \n1135. \n9. Kühl,I., Miranda,M., Posse,V ., Milenkovic,D., Mourier,A., Siira,S.J., Bonekamp,N.A., \nNeumann,U., Filipovska,A., Polosa,P .L., et al. (2016) POLRMT regulates the switch \nbetween replication primer formation and gene expression of mammalian mtDNA. \nSci Adv, 2, e1600963. \n10. Grünberg,S. and Hahn,S. (2013) Structural insights into transcription initiation by RNA \npolymerase II. Trends Biochem Sci, 38, 10.1016/j.tibs.2013.09.002. \n11. Tan,B.G., Gustafsson,C.M. and Falkenberg,M. (2023) Mechanisms and regulation of \nhuman mitochondrial transcription. Nat Rev Mol Cell Biol, 10.1038/s41580-023-\n00661-4. \n12. Shadel,G.S. and Clayton,D.A. (1993) Mitochondrial transcription initiation. Variation \nand conservation. Journal of Biological Chemistry, 268, 16083–16086. \n13. Ringel,R., Sologub,M., Morozov, Y .I., Litonin,D., Cramer,P . and Temiakov,D. (2011) \nStructure of human mitochondrial RNA polymerase. Nature, 478, 269–273. \n14. Ngo,H.B., Kaiser,J.T. and Chan,D.C. (2011) The mitochondrial transcription and \npackaging factor Tfam imposes a U-turn on mitochondrial DNA. Nat Struct Mol Biol, \n18, 1290–1296. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\n15. Hillen,H.S., Morozov, Y .I., Sarfallah,A., Temiakov,D. and Cramer,P . (2017) Structural Basis \nof Mitochondrial Transcription Initiation. Cell, 171, 1072-1081.e10. \n16. Fisher,R.P ., Topper,J.N. and Clayton,D.A. (1987) Promoter selection in human \nmitochondria involves binding of a transcription factor to orientation-independent \nupstream regulatory elements. Cell, 50, 247–258. \n17. Sologub,M., Litonin,D., Anikin,M., Mustaev,A. and Temiakov,D. (2009) TFB2 Is a \nTransient Component of the Catalytic Site of the Human Mitochondrial RNA \nPolymerase. Cell, 139, 934–944. \n18. Posse,V . and Gustafsson,C.M. (2017) Human Mitochondrial Transcription Factor B2 Is \nRequired for Promoter Melting during Initiation of Transcription. J Biol Chem, 292, \n2637–2645. \n19. Ramachandran,A., Basu,U., Sultana,S., Nandakumar,D. and Patel,S.S. (2017) Human \nmitochondrial transcription factors TFAM and TFB2M work synergistically in \npromoter melting during transcription initiation. Nucleic Acids Res, 45, 861–874. \n20. Tan,B.G., Mutti,C.D., Shi, Y ., Xie,X., Zhu,X., Silva-Pinheiro,P ., Menger,K.E., Díaz-\nMaldonado,H., Wei,W., Nicholls,T.J., et al. (2022) The human mitochondrial genome \ncontains a second light strand promoter. Molecular Cell, 82, 3646-3660.e9. \n21. Brüser,C., Keller-Findeisen,J. and Jakobs,S. (2021) The TFAM-to-mtDNA ratio defines \ninner-cellular nucleoid populations with distinct activity levels. Cell Reports, 37, \n110000. \n22. Zhu,X., Xie,X., Das,H., Tan,B.G., Shi, Y ., Al-Behadili,A., Peter,B., Motori,E., Valenzuela,S., \nPosse,V., et al. (2022) Non-coding 7S RNA inhibits transcription via mitochondrial \nRNA polymerase dimerization. Cell, 185, 2309-2323.e24. \n23. Shen,J., Goovaerts,Q., Ajjugal, Y ., De Wijngaert,B., Das,K. and Patel,S.S. (2025) Human \nmitochondrial RNA polymerase structures reveal transcription start site and \nslippage mechanism. Molecular Cell, 10.1016/j.molcel.2025.07.002. \n24. Herbine,K., Nayak,A.R., Zamudio-Ochoa,A. and Temiakov,D. (2025) Structural basis for \npromoter recognition and transcription factor binding and release in human \nmitochondria. Molecular Cell, 85, 3123-3136.e7. \n25. Posse,V ., Hoberg,E., Dierckx,A., Shahzad,S., Koolmeister,C., Larsson,N.-G., \nWilhelmsson,L.M., Hällberg,B.M. and Gustafsson,C.M. (2014) The amino terminal \nextension of mammalian mitochondrial RNA polymerase ensures promoter specific \ntranscription initiation. Nucleic Acids Res, 42, 3638–3647. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\n26. Gaspari,M., Falkenberg,M., Larsson,N.-G. and Gustafsson,C.M. (2004) The \nmitochondrial RNA polymerase contributes critically to promoter specificity in \nmammalian cells. EMBO J, 23, 4606–4614. \n27. Uchida,A., Murugesapillai,D., Kastner,M., Wang, Y ., Lodeiro,M.F ., Prabhakar,S., \nOliver,G.V ., Arnold,J.J., Maher,L.J.,III, Williams,M.C., et al. (2017) Unexpected \nsequences and structures of mtDNA required for eFicient transcription from the first \nheavy-strand promoter. eLife, 6, e27283. \n28. Morozov, Y .I. and Temiakov,D. (2016) Human Mitochondrial Transcription Initiation \nComplexes Have Similar Topology on the Light and Heavy Strand Promoters * . \nJournal of Biological Chemistry, 291, 13432–13435. \n29. Shutt,T.E., Lodeiro,M.F ., Cotney,J., Cameron,C.E. and Shadel,G.S. (2010) Core human \nmitochondrial transcription apparatus is a regulated two-component system in \nvitro. Proceedings of the National Academy of Sciences, 107, 12133–12138. \n30. Liu, Y ., Chen,Z., Wang,Z.-H., Delaney,K.M., Tang,J., Pirooznia,M., Lee,D.-Y. ,  Tu n c, I . ,  L i ,Y.  \nand Xu,H. (2022) The PPR domain of mitochondrial RNA polymerase is an \nexoribonuclease required for mtDNA replication in Drosophila melanogaster. Nat \nCell Biol, 24, 757–765. \n31. Wong,I. and Lohman,T.M. (1993) A double-filter method for nitrocellulose-filter binding: \napplication to protein-nucleic acid interactions. Proceedings of the National \nAcademy of Sciences, 90, 5428–5432. \n32. Jarmoskaite,I., AlSadhan,I., Vaidyanathan,P .P . and Herschlag,D. (2020) How to measure \nand evaluate binding aFinities. eLife, 9, e57264. \n33. Mastronarde,D.N. (2003) SerialEM: A Program for Automated Tilt Series Acquisition on \nTecnai Microscopes Using Prediction of Specimen Position. Microanal, 9, 1182–\n1183. \n34. Zheng,S.Q., Palovcak,E., Armache,J.-P ., Verba,K.A., Cheng, Y . and Agard,D.A. (2017) \nMotionCor2: anisotropic correction of beam-induced motion for improved cryo-\nelectron microscopy. Nat Methods, 14, 331–332. \n35. Zhang,K. (2016) Gctf: Real-time CTF determination and correction. Journal of Structural \nBiology, 193, 1–12. \n36. Kimanius,D., Dong,L., Sharov,G., Nakane,T. and Scheres,S.H.W. (2021) New tools for \nautomated cryo-EM single-particle analysis in RELION-4.0. Biochem J, 478, 4169–\n4185. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\n37. Zivanov,J., Nakane,T. and Scheres,S.H.W. (2020) Estimation of high-order aberrations \nand anisotropic magnification from cryo-EM data sets in RELION-3.1. IUCrJ, 7, 253–\n267. \n38. Bepler,T., Morin,A., Rapp,M., Brasch,J., Shapiro,L., Noble,A.J. and Berger,B. (2019) \nPositive-unlabeled convolutional neural networks for particle picking in cryo-\nelectron micrographs. Nat Methods, 16, 1153–1160. \n39. Punjani,A. and Fleet,D.J. (2023) 3DFlex: determining structure and motion of flexible \nproteins from cryo-EM. Nat Methods, 20, 860–870. \n40. Terwilliger,T.C., Sobolev,O.V ., Afonine,P .V . and Adams,P .D. (2018) Automated map \nsharpening by maximization of detail and connectivity. Acta Crystallogr D Struct \nBiol, 74, 545–559. \n41. Pettersen,E.F ., Goddard,T.D., Huang,C.C., Couch,G.S., Greenblatt,D.M., Meng,E.C. and \nFerrin,T.E. (2004) UCSF Chimera—A visualization system for exploratory research \nand analysis. Journal of Computational Chemistry, 25, 1605–1612. \n42. Abramson,J., Adler,J., Dunger,J., Evans,R., Green,T., Pritzel,A., Ronneberger,O., \nWillmore,L., Ballard,A.J., Bambrick,J., et al. (2024) Accurate structure prediction of \nbiomolecular interactions with AlphaFold 3. Nature, 630, 493–500. \n43. Emsley,P ., Lohkamp,B., Scott,W.G. and Cowtan,K. (2010) Features and development of \nCoot. Acta Cryst D, 66, 486–501. \n44. Adams,P .D., Afonine,P .V ., Bunkóczi,G., Chen,V .B., Davis,I.W., Echols,N., Headd,J.J., \nHung,L.-W., Kapral,G.J., Grosse-Kunstleve,R.W., et al. (2010) PHENIX: a \ncomprehensive Python-based system for macromolecular structure solution. Acta \nCrystallogr D Biol Crystallogr, 66, 213–221. \n45. Papadopoulos,J.S. and Agarwala,R. (2007) COBALT: constraint-based alignment tool for \nmultiple protein sequences. Bioinformatics, 23, 1073–1079. \n46. Sultana,S., Solotchi,M., Ramachandran,A. and Patel,S.S. (2017) Transcriptional \nfidelities of human mitochondrial POLRMT, yeast mitochondrial Rpo41, and phage \nT7 single-subunit RNA polymerases. Journal of Biological Chemistry, 292, 18145–\n18160. \n47. Schwinghammer,K., Cheung,A.C.M., Morozov, Y .I., Agaronyan,K., Temiakov,D. and \nCramer,P . (2013) Structure of human mitochondrial RNA polymerase elongation \ncomplex. Nat Struct Mol Biol, 20, 1298–1303. \n48. Zhu,X., Xie,X., Das,H., Tan,B.G., Shi, Y ., Al-Behadili,A., Peter,B., Motori,E., Valenzuela,S., \nPosse,V., et al. (2022) Non-coding 7S RNA inhibits transcription via mitochondrial \nRNA polymerase dimerization. Cell, 185, 2309-2323.e24. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\n49. Istiaq Alam,T., Kanki,T., Muta,T., Ukaji,K., Abe, Y ., Nakayama,H., Takio,K., Hamasaki,N. \nand Kang,D. (2003) Human mitochondrial DNA is packaged with TFAM. Nucleic \nAcids Res, 31, 1640–1645. \n50. Gorbea Colón,J.J., Palao,L., Chen,S.-F ., Kim,H.J., Snyder,L., Chang, Y .-W., Tsai,K.-L. and \nMurakami,K. (2023) Structural basis of a transcription pre-initiation complex on a \ndivergent promoter. Mol Cell, 83, 574-588.e11. \n51. Marcovitz,A. and Levy, Y . (2011) Frustration in protein–DNA binding influences \nconformational switching and target search kinetics. Proceedings of the National \nAcademy of Sciences, 108, 17957–17962. \n52. Tafvizi,A., Huang,F ., Fersht,A.R., Mirny,L.A. and van Oijen,A.M. (2011) A single-molecule \ncharacterization of p53 search on DNA. Proceedings of the National Academy of \nSciences, 108, 563–568. \n53. Coulombe,B. and Burton,Z.F . (1999) DNA Bending and Wrapping around RNA \nPolymerase: a “Revolutionary” Model Describing Transcriptional Mechanisms. \nMicrobiol Mol Biol Rev, 63, 457–478. \n54. Schinkel,A.H., Koerkamp,M.J.A.G., Teunissen,A.W.R.H. and Tabak,H.F . (1988) RNA \npolymerase induces DNA bending at yeast mitochondria] promoters. Nucleic Acids \nRes, 16, 9147–9163. \n55. Malarkey,C.S., Bestwick,M., Kuhlwilm,J.E., Shadel,G.S. and Churchill,M.E.A. (2012) \nTranscriptional activation by mitochondrial transcription factor A involves \npreferential distortion of promoter DNA. Nucleic Acids Res, 40, 614–624. \n56. Rubio-Cosials,A., Battistini,F ., Gansen,A., Cuppari,A., Bernadó,P ., Orozco,M., \nLangowski,J., Tóth,K. and Solà,M. (2018) Protein Flexibility and Synergy of HMG \nDomains Underlie U-Turn Bending of DNA by TFAM in Solution. Biophysical Journal, \n114, 2386–2396. \n57. Zelenaya-Troitskaya,O., Newman,S.M., Okamoto,K., Perlman,P .S. and Butow,R.A. \n(1998) Functions of the high mobility group protein, Abf2p, in mitochondrial DNA \nsegregation, recombination and copy number in Saccharomyces cerevisiae. \nGenetics, 148, 1763–1776. \n58. DiFley,J.F . and Stillman,B. (1991) A close relative of the nuclear, chromosomal high-\nmobility group protein HMG1 in yeast mitochondria. Proc Natl Acad Sci U S A, 88, \n7864–7868. \n59. DiFley,J.F . and Stillman,B. (1992) DNA binding properties of an HMG1-related protein \nfrom yeast mitochondria. Journal of Biological Chemistry, 267, 3368–3374. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nSupplementary Data \n \nFigure S1. Cryo-EM reconstruction of the mtTIC on HSP with and without TFAM, related \nto Figure 1 and 4. (A) Overview of image processing and refinement strategy. Steps \nperformed in RELION are indicated, and all other steps were performed with CryoSPARC-\n4.2. (B) Representative cryo-EM micrograph. (C) Representative 2D class averages. (D) \nGold-standard Fourier shell correlation (FSC) curve of the HSP without TFAM complex \nbetween the two half maps, with indicated resolution at FSC=0.143 shown in blue. FSC \ncurve between  the refined model and the cryo -EM map, with indicated resolution at \nFSC=0.5 shown in red. (E) The angular distribution of the particles used in the final 3D \nreconstruction of the HSP without TFAM complex. (F) Local resolution of the cryo -EM \ndensity map of the HSP without TFAM complex. (G) Gold-standard FSC curve of the HSP \nwith TFAM complex between the two half maps, with indicated resolution at FSC=0.143 \nshown in blue. FSC curve between the refined model and the cryo-EM map, with indicated \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nresolution at FSC=0.5 shown in red. ( H) The angular distribution of the particles used in \nthe final 3D reconstruction of the HSP with TFAM complex. (I) Local resolution of the cryo-\nEM density map of the HSP with TFAM complex. \n \nFigure S2. Comparison of our HSP with TFAM structure to other mtTIC structures. (A) \nConformation of PolRMT fingers domain from our HSP with TFAM structure in pink \ncompared with the LSP TIC0 structure in gray (PDB: 9MN5) (clenched) and the LSP pre-\nIC3 with incoming GTP in wheat (PDB: 9R95) (open). From the open to clenched \nconformation, the fingers domain rotates 20 º toward the active site.  Structures were \naligned by the Cα atoms of the conserved PolRMT palm domain. (B) Rotation of TFAM in \nthe mtTICs. Relative to the LSP TIC0 structure in gray (PDB: 9MN5) our HSP structure \n(orange) is rotated 12º, and 16º relative to the HSP crystal structure in pale green (PDB: \n6ERQ). Structures were aligned by the Cα atoms of the conserved PolRMT and TFB2M \ncore. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\n \n \nFigure S3. Trajectory of upstream DNA in the HSP with and without TFAM complexes. \n(A) In the HSP with TFAM complex, TFAM bends the upstream DNA proximal to the active \nsite into a U-turn shape and the more distal upstream DNA extends linearly after the bend. \n(B) In the HSP without TFAM complex, only a short portion of the upstream DNA is \nobserved, which is linear in the absence of TFAM-induced bending. \n \nFigure S4. Electrostatic interactions of the PolRMT tether helix in the HSP with or without \nTFAM complexes. (A) Electrostatic surface of TFAM and cartoon representation of the  \nPolRMT tether helix in the HSP with TFAM complex. The highly positively charged surface \nof the tether helix engages the negatively charged helix 3 of the HMG -B box. (B) \nElectrostatic surface of the positively charged PolRMT tether helix near the negatively \ncharged DNA backbone in the HSP without TFAM complex. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\n \nFigure S5. The auto-inhibitory effect of the PolRMT tether helix follows a similar but less \npronounced trend at higher PolRMT concentration, related to Figure 4 . Transcription \nreactions contained 20 nM DNA template, 100 nM TFB2M, 0 or 60 nM TFAM, and 0 or \n300 nM PolRMT. (A) Quantification of transcript production from the NS DNA substrate. \n(B) Quantification of transcript production from the LSP. (C) Quantification of transcript \nproduction from the HSP. (D) Quantification of transcript production from the LSP2. Mean \nand SEM were determined from 3 independent replicates. Significance was determined \nusing a 1-way ANOVA test with Tukey’s multiple comparisons test. Significance: * p<0.05, \n** p<0.01, **** p<0.0001. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\n \nFigure S6. Sequence and charge conservation of the PolRMT tether helix and TFAM \nHMG-B box helix 3. (A) Alignment of PolRMT and its homologs from various eukaryotic \nspecies. Red shading indicates high sequence conservation, blue shading indicates lower \nsequence conservation, and black outline boxes indicate location of probable tether helix \nsequences. (B) Sequences of the prob able tether helix sequences with blue boxes \nindicating the positively charged residues, * indicates sequences that did not align well \nwith the human PolRMT tether helix. (C) Alignment of TFAM and its homologs from \nmetazoan species that utilize TFAM as part of a three -component mtTIC. Red shading \nindicates high sequence conservation, blue shading indicates lower sequence \nconservation, and black outline boxes indicate the HMG-B box helix 3. (D) Sequences of \nthe TFAM HMG-B box helix 3 with red boxes indicating the negatively charged residues. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\n \nTable S1. Oligonucleotides for PolRMT cloning and in vitro transcription templates. \nOligo \nName \nDescription Sequence (5' to 3') \nHSP 60 \nNT \nHSP NT -60 to \n+11, mutated \nfrom -4 to +3 to \ncreate bubble \nCCATCCTACCCAGCACACACACACCGCTGCTAACC\nCCATACCCCGAACCAACCAAATTATCCCGACACCCC  \nHSP 60 \nTS \nHSP TS -60 to \n+11 \nGGGGTGTCTTTGGGGTTTGGTTGGTTCGGGGTATG\nGGGTTAGCAGCGGTGTGTGTGTGCTGGGTAGGATG\nG  \nLSP 40 \nNT \nLSP NT -40 to \n+20 \nGTGTTAGTTGGGGGGTGACTGTTAAAAGTGCATACC\nGCCAAAAGATAAAATTTGAAATCT \nLSP 40 \nTS \nLSP TS -40 to \n+20 \nAGATTTCAAATTTTATCTTTTGGCGGTATGCACTTTTA\nACAGTCACCCCCCAACTAACAC \nLSP 70 \nNT \nLSP NT -70 to \n+20 \nAGTAGTATGGGAGTGGGAGGGGAAAATAATGTGTTA\nGTTGGGGGGTGACTGTTAAAAGTGCATACCGCCAA\nAAGATAAAATTTGAAATCT \nLSP 70 \nTS \nLSP TS -70 to \n+20 \nAGATTTCAAATTTTATCTTTTGGCGGTATGCACTTTTA\nACAGTCACCCCCCAACTAACACATTATTTTCCCCTC\nCCACTCCCATACTACT \nHSP 40 \nNT \nHSP NT -40 to \n+20 \nACACCGCTGCTAACCCCATACCCCGAACCAACCAAA\nCCCCAAAGACACCCCCCACAGTTT \nHSP 40 \nTS \nHSP TS -40 to \n+20 \nAAACTGTGGGGGGTGTCTTTGGGGTTTGGTTGGTT\nCGGGGTATGGGGTTAGCAGCGGTGT \nHSP 70 \nNT \nHSP NT -70 to \n+20 \nCAACCCCCGCCCATCCTACCCAGCACACACACACC\nGCTGCTAACCCCATACCCCGAACCAACCAAACCCC\nAAAGACACCCCCCACAGTTT \nHSP 70 \nTS  \nHSP TS -70 to \n+20 \nAAACTGTGGGGGGTGTCTTTGGGGTTTGGTTGGTT\nCGGGGTATGGGGTTAGCAGCGGTGTGTGTGTGCTG\nGGTAGGATGGGCGGGGGTTG \nLSP2 40 \nNT \nLSP2 NT -40 to \n+20 \nGAGTCAATACTTGGGTGGTACCCAAATCTGCTTCCC\nCATGAAAGAACAGAGAATAGTTTA \nLSP2 40 \nTS \nLSP2 TS -40 to \n+20 \nTAAACTATTCTCTGTTCTTTCATGGGGAAGCAGATTT\nGGGTACCACCCAAGTATTGACTC \nLSP2 70 \nNT \nLSP2 NT -70 to \n+20 \nGTACGAAATACATAGCGGTTGTTGATGGGTGAGTCA\nATACTTGGGTGGTACCCAAATCTGCTTCCCCATGAA\nAGAACAGAGAATAGTTTA \nLSP2 70 \nTS \nLSP2 TS -70 to \n+20 \nTAAACTATTCTCTGTTCTTTCATGGGGAAGCAGATTT\nGGGTACCACCCAAGTATTGACTCACCCATCAACAAC\nCGCTATGTATTTCGTAC \nNS 70 \nNT \nNS NT -70 to \n+20 \nTATATGATATGTCTCCATACCCATTACAATCTCCAGCA\nTTCCCCCTCAAACCTAAGAAATATGTCTGATAAAAGA\nGTTACTTTGATAGAG \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nmtDNA \nsequence 4211-\n4300 \n \nNS 70 \nTS \nNS TS -70 to \n+20 \nmtDNA \nsequence 4211-\n4300 \nCTCTATCAAAGTAACTCTTTTATCAGACATATTTCTTA\nGGTTTGAGGGGGAATGCTGGAGATTGTAATGGGTAT\nGGAGACATATCATATA \n \nES 42 \nNT \nNT -42 to +10 \nwith -9 to -1 \nbubble for \nelongation \nGCACTCACAGTCGCATCATAATCGCTCAACGATTAAT\nTATACACGTAGCC \n \nES 42 \nTS \nTS -42 to +10 \nfor elongation \nGGCTACGTGTCGCCGGGCGCGTTGAGCGATTATGA\nTGCGACTGTGAGTGC \n \nES 72 \nNT \nNT -72 to +10 \nwith -9 to -1 \nbubble for \nelongation \nACTATTCTGCCTAGCAAACTCAAACTACGAACGCAC\nTCACAGTCGCATCATAATCGCTCAACGATTAATTATA\nCACGTAGCC \n \nES 72 \nTS \nTS -72 to +10 \nfor elongation \nGGCTACGTGTCGCCGGGCGCGTTGAGCGATTATGA\nTGCGACTGTGAGTGCGTTCGTAGTTTGAGTTTGCTA\nGGCAGAATAGT \n \nES RNA FAM-labeled \nRNA pairs with \nthe TS from -9 \nto -1 with a 3-nt \noverhang on the \n5' end \n/56-FAM/ rUrUrUrCrGrCrCrCrGrGrCrG \n \nPolRMT \n3KE \nFwd \nPolRMT \nMutation K425E, \nK428E, K432E  \nAAGTGGAGCATGCACGCGAGACCCTGAAAACCTTA\nCGTGATCAGT \nPolRMT \n3KE Rev \nPolRMT \nMutation K425E, \nK428E, K432E \nGTGCATGCTCCACTTCCTCACTCGGCAGGGTCGGT\nTT \nPolRMT \nΔTH \nFwd \nPolRMT \ntruncation of \nresidues 122-\n146 \nCCGTTTCAGAGTGGCG \n \nPolRMT \nΔTH \nRev \nPolRMT \ntruncation of \nresidues 122-\n146 \nACGACCACACGGCAC \n \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nTable S2. Structure alignment of the HSP with TFAM TIC core to other mtTIC structures. \nStructures were aligned by the Cα atoms of the conserved PolRMT and TFB2M core. \nPDB ID Description Aligned Atoms \n(#) \nRMSD (Å) Rotation of \nTFAM (º) \n6ERQ HSP TIC0 \ncrystal structure \n1106 1.135 15.9 \n6ERP LSP TIC0 crystal \nstructure \n1110 1.108 16.7 \n9MN5 LSP IC0 893 0.857 11.8 \n9MN4 LSP IC3 with 3-\nnt RNA  \n850 0.895 14.1 \n9GZM LSP pre-IC3 with \n2-nt RNA and \nincoming GTP \n1159 0.856 7.2 \n9R95 LSP slipped IC3 \nwith 2-nt RNA, \nATP, and \nincoming GTP \n1136 0.716 6.4 \n9R96 LSP slipped pre-\nIC4 with 2-nt \nRNA, dATP, and \nincoming GTP \n1149 0.837 5.7 \n9GZN LSP pre-IC3 with \n2-nt RNA, \nincoming GTP, \nand no TFAM \n1140 0.901 N/A \n9GZO LSP slipped IC3 \nwith 2-nt RNA, \nATP, incoming \nGTP, and no \nTFAM \n1136 1.023 N/A \n11PS HSP without \nTFAM \n1331 0.601 N/A \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint \n\nTable S3 . Structure alignment of the HSP without TFAM TIC core to other mtTIC \nstructures. Structures were aligned by the C α atoms of the conserved PolRMT and \nTFB2M core. \nPDB ID Description Aligned Atoms (#) RMSD (Å) \n6ERQ HSP TIC0 crystal \nstructure \n1117 1.290 \n6ERP LSP TIC0 crystal \nstructure \n1118 1.259 \n9MN5 LSP IC0  889 0.912 \n9MN4 LSP IC3 with 3-nt \nRNA  \n836 1.011 \n9GZM LSP pre-IC3 with 2-\nnt RNA and \nincoming GTP \n1185 1.187 \n9R95 LSP slipped IC3 \nwith 2-nt RNA, ATP, \nand incoming GTP \n1181 1.074 \n9R96 LSP slipped pre-\nIC4 with 2-nt RNA, \ndATP, and incoming \nGTP \n1181 1.148 \n9GZN LSP pre-IC3 with 2-\nnt RNA, incoming \nGTP, and no TFAM \n1180 1.201 \n9GZO LSP slipped IC3 \nwith 2-nt RNA, ATP, \nincoming GTP, and \nno TFAM \n1159 1.244 \n11PR HSP with TFAM 1331 0.601 \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted April 12, 2026. ; https://doi.org/10.64898/2026.04.09.717523doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}