REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains
BL21(DE3)	Sigma-Aldrich	Cat. #CMC0014
BL21(DE3) Codon Plus (RIL)	Agilent	Cat. # 230245
Chemicals, peptides, and recombinant proteins
Protein Thermal Shift Dye for protein thermal shift assay	Applied Biosystems	Cat. # 4461146
Uridine 5′(α-thio) triphosphate	Jenna Bioscience	Cat. # NU-411S
Acrylamide:bis-acrylamide solution	Bio-Rad Laboratories	Cat. # 161-0154
2× Laemmli sample buffer	Bio-Rad Laboratories	Cat. # 161-0737
Urea	Sigma-Aldrich	Cat. # U5378
Ammonium persulfate	Sigma-Aldrich	Cat. # A3678
TEMED	Sigma-Aldrich	Cat. # T9281
ATP, GTP, and UTP	Thermo Scientific	Cat. # R0481
3′-dCTP	TriLink BioTechnologies	Cat. # N-3003
α-32P-GTP	PerkinElmer	Cat. # BLU506H250UC
Poly(ethyleneimine), average Mw ˜1300 Da, 50% w/v	Sigma	Cat. # 482595-100ML
Remel™ LB Agar, Miller	Thermo Fisher Scientific	Cat. # R453632
Plasmid pRK793 for expressing tobacco etch virus (TEV) protease	Kapust et al., 2001	Addgene.org plasmid # 8827
Deposited data
Yeast mitochondrial ΔN100 y-mtRNAP /MTF1/dsDNA, pre-initiation complex	This paper	PDB 6YMV
Cryo-EM map for above	This paper	EMD-10845
Yeast mitochondrial ΔN100 y-mtRNAP /MTF1/dsDNA/RNA/UTPαS, initiation complex	This paper	PDB 6YMW
Cryo-EM map for above	This paper	EMD-10846
Original source data	This paper	https://dx.doi.org/10.17632/vtd45jkx6v.3
Oligonucleotides
Modified yeast mitochondrial 15S promoter nontemplate strand 5′ -CGAATAAGTATTGATATAAGTAATAGATAATGC	IDT	N/A
Modified yeast mitochondrial 15S promoter template 5′ -GCATTATCTACCGACAATATCAATACTTATTCG	IDT	N/A
Yeast mitochondrial 21S rRNA promoter template 5′ -GGTATTTCAAATCTATTATTCTACTTTTTACTACTT ATATATATAATAATAATAATA	IDT	N/A
Yeast mitochondrial 21S rRNA promoter nontemplate 5′ -TATTATTATTATTATATATATAAGTAG TAAAAAGTAGAATAATAGATTTGAAATACC	IDT	N/A
RNA sequence (primer strand): 5′ pppGpG 3′	TriLink BioTechnologies	Cat. # O-31011
Recombinant DNA
Plasmid pTrcHisC for expressing yeast MTF1	Tang et al., 2009	N/A
Plasmid ProEXHTb for expressing yeast ΔN100 y-mtRNAP	Paratkar et al., 2011	N/A
Software and algorithms
PHENIX 1.16	Liebschner et al., 2019	phenix-online.org
RELION 3.0.8	Zivanov et al., 2018	https://www3.mrc-lmb.cam.ac.uk/relion/index.php/Main_Page
CTFFIND-4	Rohou and Grigorieff, 2015	https://grigoriefflab.umassmed.edu/ctffind4
Scipion	de la Rosa-Trevin et al., 2016	http://scipion.i2pc.es
Curves+	(Blanchet et al., 2011)	https://bisi.ibcp.fr/tools/curves_plus/index.html
MotionCor2 (included in RELION 3.0.8)	(Zheng et al., 2017)	https://emcore.ucsf.edu/ucsf-motioncor2
Coot 0.8.2	Emsley and Cowtan, 2004	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
PyMOL	Schrödinger, LLC.	https://pymol.org/2/
Chimera	(Pettersen et al., 2004)	https://www.cgl.ucsf.edu/chimera/download.html
DYNAMICS 7.10.0.23	Wyatt Technology	N/A
ResMap	Kucukelbir et al., 2014	N/A
Other
Titan Krios 300 keV cryo-electron microscope	FEI/Thermo Fisher	N/A
Titan Glacios 200 keV cryo-electron microscope	FEI/Thermo Fisher	N/A
PELCO easiGlow Glow Discharge Cleaning System	Ted Pella	N/A
EM GP grid plunger	Leica	N/A
Quantifoil® R 1.2/1.3 200 Cu	Quantifoil	Q42272
WhatmanTM filter paper, grade 1	GE Healthcare	1001-055
Äkta pure 25 L1	GE Healthcare	29018225
miniDAWN TREOS	Wyatt Technology	N/A
DynaPro NanoStar DLS	Wyatt Technology	N/A
Optilab T-rEX	Wyatt Technology	N/A
NanoDropTM One UV-VIS Spectrophotometer	Thermo Fisher	N/A
Amicon® Ultra-4 10K centrifugal filter	Merck Millipore	UFC801024
QuantStudio5 qPCR	Thermo Fisher	A28139
Superdex 200 Increase 10/300 GL	GE Healthcare	28990944
HiTrap DEAE FF column (5 mL)	Cytiva	17515401
HisTrap HP His tag protein purification column (5 mL)	Cytiva	17524801
HiTrap Heparin HP affinity column (1 mL)	Cytiva	17040601
Mini dry bath	Greiner Bio-One	848060
Microcon 100 kDa with Biomax membrane	Millipore Sigma	MPE100025