KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Bacterial and Virus Strains | ||
| E. coli BL21(DE3) | EMD Millipore | |
| Chemicals, Peptides, and Recombinant Proteins | ||
| 3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO) | Anatrace | Cat# C317 |
| E. coli RNAP (cryo-EM samples) | Chen et al., 2019b | N/A |
| E. coli RNAP, WT (Biochemistry) | Svetlov and Artsimovitch, 2015 | |
| E. coli RNAP, βA474L | This paper | |
| E. coli RNAP, βA474V | This paper | |
| E. coli RNAP, βR378A | This paper | |
| E. coli RNAP, βR378E | This paper | |
| E. coli RNAP, βR378E βR394E | This paper | |
| E. coli RNAP, βR394A | This paper | |
| E. coli RNAP, βR394E | This paper | |
| Polyethyleneimine | Fisher Scientific | Cat# AC178572500 |
| Deposited Data | ||
| Coordinates of E. coli Eσ70 | Chen et al., 2019b | PDB: 6P1K |
| Coordinates of E. coli Eσ70/Wt rpsT P2 | Chen et al., 2019b | PDB: 6OUL |
| Coordinates of E. coli TraR/Eσ70 (I) | Chen et al., 2019b | PDB: 6N57 |
| Coordinates of E. coli TraR/Eσ70 (II) | Chen et al., 2019b | PDB: 6N58 |
| Coordinates of E. coli TraR/Eσ70/Wt rpsT P2 (T-preRPo) | This paper | PDB: 6PSV |
| Coordinates of E. coli TraR/Eσ70/Wt rpsT P2 (T-RPc) | This paper | PDB: 6PSQ |
| Coordinates of E. coli TraR/Eσ70/Wt rpsT P2 (T-RPi1) | This paper | PDB: 6PSR |
| Coordinates of E. coli TraR/Eσ70/Wt rpsT P2 (T-RPi2) | This paper | PDB: 6PSU |
| Coordinates of E. coli TraR/Eσ70/Wt rpsT P2 (T-RPo) | This paper | PDB: 6PSW |
| Coordinates of E. coli TraR/Eσ70/Wt rpsT P2* (T-RPi1.5a) | This paper | PDB: 6PSS |
| Coordinates of E. coli TraR/Eσ70/Wt rpsT P2* (T-RPi1.5b) | This paper | PDB: 6PST |
| Cryo-EM map of E. coli TraR/Eσ70/rpsT P2* (T-RPi1.5a) | This paper | EMD_20462 |
| Cryo-EM map of E. coli TraR/Eσ70/Wt rpsT P2 (T-preRPo) | This paper | EMD-20465 |
| Cryo-EM map of E. coli TraR/Eσ70/Wt rpsT P2 (T-RPc) | This paper | EMD-20460 |
| Cryo-EM map of E. coli TraR/Eσ70/Wt rpsT P2 (T-RPi1) | This paper | EMD-20461 |
| Cryo-EM map of E. coli TraR/Eσ70/Wt rpsT P2 (T-RPi2) | This paper | EMD-20464 |
| Cryo-EM map of E. coli TraR/Eσ70/Wt rpsT P2 (T-RPo) | This paper | EMD-20466 |
| Cryo-EM map of E. coli TraR/Eσ70/Wt rpsT P2* (T-RPi1.5b) | This paper | EMD-20463 |
| Experimental Models: Organisms/Strains | ||
| Escherichia coli | ||
| Oligonucleotides | ||
| pIA900 βA474L: GTGTAGAGCGTCTGGTGAAAGAGCGTC | This paper, IDT | |
| pIA900 βA474V: GTGTAGAGCGTGTGGTGAAAGAGCGTC | This paper, IDT | |
| pIA900 βR378A: GAGCCGCCGACTGCCGAAGCAGCTGAAAGCCTG | This paper, IDT | |
| pIA900 βR378E: GAGCCGCCGACTGAAGAAGCAGCTGAAAGCCTG | This paper, IDT | |
| pIA900 βR394A: CTTCTCCGAAGACGCTTATGACTTGTCTGC | This paper, IDT | |
| pIA900 βR394E: CTTCTCCGAAGACGAATATGACTTGTCTGC | This paper, IDT | |
| rpsTP2(−60to+25)_−11/−10CG_bot: 5’-GCG TTC TAT ATG GAC AAT TCA AAG GCC GAG GAA TGC GCC CTT TTA GCC TTC TTT TGT CAA TGG ATT TGT GCA AAT AAG CGC CGC C-3’ |
This paper, IDT | |
| rpsTP2(−60to+25)_(T-7A)_top: 5’-GGC GGC GCT TAT TTG CAC AAA TCC ATT GAC AAA AGA AGG CTA AAA GGG CAT ATA CCT CGG CCT TTG AAT TGT CCA TAT AGA ACG C-3’ |
This paper, IDT | |
| rpsTP2(−60to+25)_bot: 5’-GCG TTC TAT ATG GAC AAT TCA AAG GCC GAG GAA TAT GCC CTT TTA GCC TTC TTT TGT CAA TGG ATT TGT GCA AAT AAG CGC CGC C-3’ |
This paper, IDT | |
| rpsTP2(−60to+25)_top: 5’-GGC GGC GCT TAT TTG CAC AAA TCC ATT GAC AAA AGA AGG CTA AAA GGG CAT ATT CCT CGG CCT TTG AAT TGT CCA TAT AGA ACG C-3’ |
This paper, IDT | |
| Recombinant DNA | ||
| E. coli RNAP, pIA900 WT | Svetlov and Artsimovitch, 2015 | |
| E. coli RNAP, pIA900 βA474L | This paper | pRLG15445 |
| E. coli RNAP, pIA900 βA474V | This paper | pRLG15444 |
| E. coli RNAP, pIA900 βR378A | This paper | pRLG15446 |
| E. coli RNAP, pIA900 βR378E | This paper | pRLG15447 |
| E. coli RNAP, pIA900 βR378E βR394E | This paper | pRLG15450 |
| E. coli RNAP, pIA900 βR394A | This paper | pRLG15448 |
| E. coli RNAP, pIA900 βR394E | This paper | pRLG15449 |
| p770 | Ross, et al., 1990 | pRLG770 |
| p770-rpsT P2 (−89 to +50) | Lemke et al., 2011 | pRLG14658 |
| p770-rrnB P1 (−88 to +50) | Ross et al., 2016 | pRLG13065 |
| p770-thrABC (−72 to +16) | Barker, 2001 | pRLG15276 |
| pACYCDuet-1_Ec_rpoZ | Twist et al., 2011 | |
| pEcrpoABC(-XH)Z | Twist et al., 2011 | |
| pET28a | EMD Millipore | |
| pET28a-His10-SUMO rpoD | Chen et al., 2017 | |
| pET28a-His10-SUMO traR | Chen et al. 2019b | pRLG15142 |
| pIA900, multisubunit RNAP plasmid | Svetlov and Artsimovitch, 2015 | |
| pSL6-rpsT P2 (−68 to +50) | Gopalkrishnan et al., 2017 | pRLG11272 |
| pSL6-rpsT P2 (−68 to +50) (T-7A) | This paper | pRLG12844 |
| Software and Algorithms | ||
| Bayesian Polishing | Zivanov et al., 2018 | https://github.com/3dem/relion |
| blocfilt | Cardone et al., 2013 | https://lsbr.niams.nih.gov/bsoft/programs/blocres.html |
| blocres | Cardone et al., 2013 | https://lsbr.niams.nih.gov/bsoft/programs/blocres.html |
| Coot | Emsley and Cowtan, 2004 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot |
| cryoSPARC | Punjani et al., 2017 | https://cryosparc.com/ |
| CTFFIND4 | Rohou and Grigorieff, 2015 | http://grigoriefflab.janelia.org/ctffind4 |
| Gautomatch | N/A | http://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/ |
| Gctf | Zhang, 2016 | https://www.mrc-lmb.cam.ac.uk/kzhang/Gctf/ |
| GraphPad Prism | GraphPad | https://www.graphpad.com/scientific-software/prism |
| ImageQuant 5.2 | GE Healthcare, Pittsburgh PA | |
| m/z- Knexus edition | Proteometrics, LLC | |
| Molprobity | Chen et al., 2010 | http://molprobity.biochem.duke.edu |
| MotionCor2 | Zheng et al., 2017 | |
| MTRIAGE | Afonine et al., 2018 | https://www.phenix-online.org/documentation/reference/mtriage.html |
| PDBePISA | Krissinel and Henrick, 2007 | https://www.ebi.ac.uk/pdbe/pisa/ |
| PHENIX | Adams et al., 2010 | https://www.phenix-online.org/documentation/index.html |
| Qual Browser Thermo Xcalibur version 3.0.63 | Thermo Fisher Scientific Inc. | Thermo Scientific MS instruments |
| RELION | Scheres, 2012 | https://github.com/3dem/relion |
| SBGrid | Morin et al., 2013 | https://sbgrid.org/ |
| SerialEM | Mastronarde, 2005 | http://bio3d.colorado.edu/SerialEM |
| The PyMOL Molecular Graphics System | Schrödinger, LLC | http://www.pymol.org |
| UCSF Chimera | Pettersen et al., 2004 | https://www.cgl.ucsf.edu/chimera |
| UniDec version 3.2 | Marty et. al., 2015 | https://github.com/michaelmarty/UniDec/releases |
| Other | ||
| Bio-Rex 70 cation exchange resin, analytical grade, 100–200 mesh | Bio-Rad | Cat# 1425842 |
| C-flat CF-1.2/1.3 400 mesh gold grids | Electron Microscopy Sciences | Cat# CF413–100-Au |
| HiLoad 26/600 Superdex 200 pg | GE Healthcare Life Sciences | Cat# 28989336 |
| HiTrap IMAC HP | GE Healthcare Life Sciences | Cat# 17092003 |
| Isotope [α−32P]UTP | Perkin Elmer | Cat # BLU507H500UCI |
| Isotope [α32P]-dCTP | Perkin Elmer | Cat # BLU013H250UCI |
| Superose 6 INCREASE 10/300 GL | GE Healthcare Life Sciences | Cat# 29091596 |
| Zeba Micro Spin Desalting Columns, 40K MWCO | Thermo Pierce | Cat. # 87765 |