A trailing ribosome speeds up RNA polymerase at the expense of transcript fidelity via force and allostery

. Author manuscript; available in PMC: 2023 Apr 27.

Published in final edited form as: Cell. 2023 Mar 16;186(6):1244–1262.e34. doi: 10.1016/j.cell.2023.02.008

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Purified anti-E. coli RNA polymerase $α$ antibody [4RA2], monoclonal	BioLegend	Cat#663102; RRID:AB_2564409
Bacterial and virus strains
E. coli BL21 λDE3	Lab stock	N/A
E. coli BL21 λDE3 pLysS (CamR)	Novogen	Cat#69451
HB101	Coli Genetic Stock Center at Yale	Cat#12554
MRE600	Lab stock	N/A
MG1655	Coli Genetic Stock Center at Yale	Cat#CGSC6300
Rosetta(DE3) pLysS	Sigma-Aldrich	Cat#70956
$Δ ssrA$ (MG1655 smpB_ssrA::kanR)	Saito et al.,2022¹¹⁶	SKEC114
$Δ smrB$ (MG1655 smrB::camR)	Saito et al.,2022¹¹⁶	SKEC120
$Δ ssrA$ $Δ smrB$ (MG1655 smpB_ssrA::kanR smrB::camR)	Saito et al.,2022¹¹⁶	SKEC121
$Δ smrA$ (MG1655 smrA::camR)	Saito et al.,2022¹¹⁶	SKEC123
Chemicals, peptides, and recombinant proteins
2-mercaptoethano	Sigma-Aldrich	Cat#M3148; CAS: 60-24-2
5′ Deadenylase	New England Biolabs	Cat#M0331S
¹⁰N-Formyltetrahydrofolate	Biosynth^® Carbosynth	Cat#FF165438; CAS: 2800-34-2
$α$ -Lactose monohydrate	Sigma-Aldrich	Cat#L3625; CAS: 5989-81-1
Ammonium acetate, CH₃CO₂NH4	Thermo Fisher Scientific	Cat#BP3261; CAS: 631-61-8
Ammonium chloride, NH₄Cl	Sigma-Aldrich	Cat#09718; CAS: 12125-02-9
Ammonium sulfate, (NH₄)₂SO₄	Sigma-Aldrich	Cat#A4418; CAS: 7783-20-2
Ampicillin	Sigma-Aldrich	Cat#A0166; CAS: 69-53-4
Apyrase	New England Biolabs	Cat#M0393S
Ascorbic acid	Sigma-Aldrich	Cat#A5960; CAS: 50-81-7
ATP, [α−³²P]	Perkin Elmer	Cat#BLU003H250U C
ATP, [γ−³²P]	Perkin Elmer	Cat#NEG035C005M C
Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid), ABTS	Pierce	Cat#34026; CAS: 30931-67-0
Benzonase	Millipore	Cat#70746; CAS: 9025-65-4
BsaI-HF^®	New England Biolabs	Cat#R3535L
Calcium chloride dihydrate	Sigma-Aldrich	Vat#C5080; CAS: 10035-04-8
Chloramphenicol	Sigma-Aldrich	Cat#C0378; CAS: 56-75-7
Chloroform	Sigma-Aldrich	Cat#C2432; CAS: 67-66-3
cOmpleteTM, EDTA-free protease inhibitor cocktail	Roche	Cat#11873580001
Creatine phosphate	Roche	Cat# 10621714001; CAS: 71519-72-7
Creatine Phosphokinase from rabbit muscle	Sigma-Aldrich	Cat#C3755
Cycloheximide	Sigma-Aldrich	Cat#C7698; CAS: 66-81-9
D-Biotin	Thermo Fisher Scientific	Cat#B20656; CAS: 58-85-5
Dithiothreitol, C₄H₁₀O₂S₂	Sigma-Aldrich	Cat#43815; CAS: 3483-12-3
Dimethyl sulfoxide, anhydrous	Sigma-Aldrich	Cat#276855; CAS: 67-68-5
E. coli RNA Polymerase, Core Enzyme	New England Biolabs	Cat#M0550S
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDC	Thermo Fisher Scientific	Cat#22980; CAS: 25952-53-8
Ethanol, 200-proof	Koptec	Cat#V1016; CAS: 64-17-5
Ethylenediaminetetraacetic acid, EDTA	Sigma-Aldrich	Cat#E6758; CAS: 60-00-4
Exonuclease I	Thermo Fisher Scientific	Cat#EN0581
Exonuclease III	Thermo Fisher Scientific	Cat#EN0191
Fidaxomicin	Apexbio Technology LLC.	Cat#B175550; CAS: 873857-62-6
Formamide (Deionized)	Sigma-Aldrich	Cat#F9037; CAS: 75-12-7
Fusidic acid	Sigma-Aldrich	Cat#F0756; CAS: 6990-06-3
GGGGDGDY-Lys(biotin)	Genscript	NA
Glucose, C₆H₁₂O₆	Sigma-Aldrich	Cat#G8270; CAS: 50-99-7
Glycerol	Sigma-Aldrich	Cat#G5516; CAS: 56-81-5
Glycine	Sigma-Aldrich	Cat#50046; CAS: 56-40-6
Glycogen from mussels	Roche	Cat#10901393001; CAS: 9005-79-2
Guanosine 5′-diphosphate (GDP) sodium salt	Sigma-Aldrich	Cat#G7127; CAS: 43139-22-6
HEPES	Sigma-Aldrich	Cat# H4034; CAS: 7365-45-9
Horseradish peroxidase	Pierce	Cat#31490; CAS: 9003-99-0
Imidazole	Sigma-Aldrich	Cat#I5513; CAS: 288-32-4
Isopropyl $β$ -D-1-thiogalactopyranoside, IPTG	Thermo Fisher Scientific	Cat#50-490-794; CAS: 367-93-1
Kanamycin sulfate	Sigma-Aldrich	Cat#60615; CAS: 70560-51-9
Lambda Phage DNA	New England Biolabs	Cat#N3011S; CAS: 91080-14-7
Magnesium acetate tetrahydrate, (CH3COO)₂Mg•4H₂O	Sigma-Aldrich	Cat#M5661; CAS: 16674-78-5
Magnesium chloride, MgCl₂	Thermo Fisher Scientific	Cat# BP214; CAS: 7786-30-3
Magnesium sulfate, MgSO₄	Sigma-Aldrich	Cat#M7506; CAS: 7487-88-9
MES hydrate	Sigma-Aldrich	Cat#M8250; CAS: 1266615-59-1
Myokinase from rabbit muscle	Sigma-Aldrich	Cat#M3003; CAS: 9013-02-9
Mueller Hinton Agar	Sigma-Aldrich	Cat#70191-100G
Phenol	Sigma-Aldrich	Cat#P4557; CAS: 108-95-2
Phenol:Chloroform:Isoamyl Alcohol 25:24:1	Sigma-Aldrich	Cat#P2069
Phosphocreatine	Sigma-Aldrich	Cat#P1937; CAS: 108321-17-1
Phusion HotStart II High Fidelity DNA Polymerase	Thermo Fisher Scientific	Cat#F537S
Phusion^® High-Fidelity DNA Polymerase	New England Biolabs	Cat#M0530S
Pierce^™ NeutrAvidin Protein	Thermo Fisher Scientific	Cat#31000
Potassium acetate, KOAc	Thermo Fisher Scientific	Cat#BP364; CAS: 127-08-2
Potassium chloride, KCl	Sigma-Aldrich	Cat#P9541; CAS: 7447-40-7
Potassium hydroxide, KOH	Thermo Fisher Scientific	Cat#P250-1; CAS: 1310-58-3
Potassium phosphate dibasic, K₂HPO₄	Sigma-Aldrich	Cat#60353; CAS: 7758-11-4
Potassium phosphate monobasic, KH₂PO₄	Sigma-Aldrich	Cat#P9791; CAS: 7778-77-0
Potassium pyrophosphate, K₄P₂O₇	Sigma-Aldrich	Cat#322431; CAS: 7320-34-5
Pseudouridimycin	AdipoGen	Cat# AGCN20316M005; CAS: 1566586-52-4
Putrescine dihydrochloride, NH₂(CH₂)₄NH₂•2HCl	Sigma-Aldrich	Cat#P5780; CAS: 333-93-7
Pyrophosphatase, Inorganic from baker’s yeast (S. cerevisiae)	Sigma-Aldrich	Cat#I1891; CAS: 9024-82-2
RecJ_f	New England Biolabs	Cat#M0264S
RNaseOUT^™ Recombinant Ribonuclease Inhibitor	Thermo Fisher Scientific	Cat#10777
rNTPs	Promega	Cat#E6000
SequaGel UreaGel 29:1 Concentrate	National Diagnostics	Cat#EC-828
Sodium azide	Sigma-Aldrich	Cat#71289; CAS: 26628-22-8
Sodium chloride, NaCl	Sigma-Aldrich	Cat#S9888; CAS: 7647-14-5
Sodium dodecyl sulfate, SDS	Sigma-Aldrich	Cat#L5750; CAS: 151-21-3
Sodium phosphate dibasic, Na₂HPO₄	Sigma-Aldrich	Cat#S3264; CAS: 7558-79-4
Spermidine trihydrochloride	Sigma-Aldrich	Cat#85578; CAS: 334-50-9
Sucrose	Sigma-Aldrich	Cat#S5016; CAS: 57-50-1
SuperScript^™ IV Reverse Transcriptase	Thermo Fisher Scientific	Cat#18090010
SYBR Gold Nucleic Acid Gel Stain	Thermo Fisher Scientific	Cat#S-11494
T4 DNA Ligase (2,000,000 units/ml)	New England Biolabs	Cat#M0202T
T4 DNA Ligase (400,000 units/ml)	New England Biolabs	Cat#M0202S
T4 Polynucleotide Kinase	New England Biolabs	Cat#M0201S
T4 RNA Ligase 2, truncated K227Q	New England Biolabs	Cat#M0351S
Terrific Broth	Thermo Fisher Scientific	Cat#BP2468500
Tetracycline hydrochloride	Sigma-Aldrich	Cat#T7660; CAS: 64-75-5
Total tRNA from MRE600	Roche	Cat#10109541001
Tris(2-carboxyethyl)phosphine hydrochloride, TCEP	Sigma-Aldrich	Cat#646547; CAS: 51805-45-9
Tris Base	Genesee Scientific	Cat#18-146; CAS: 77-86-1
tRNA^fMet	tRNAprobes	Cat#FM-03
Trolox	Sigma-Aldrich	Cat#238813; CAS: 53188-07-1
Tryptone (Gibco^™ Bacto^™)	Thermo Fisher Scientific	Cat#DF0123-17-3; CVAS: 91079-40-2
TWEEN^® 20	Sigma-Aldrich	Cat#P9416; CAS: 9005-64-5
Ultra Low Range DNA Marker	Thermo Fisher Scientific	Cat#SM1213
Yeast extract	Sigma-Aldrich	Cat#Y1625; CAS: 8013-01-2
Critical commercial assays
5′ DNA Adenylation Kit	New England Biolabs	Cat#E2610S
L-Amino acids	Sigma-Aldrich	Cat#LAA21
MEGAscript^® T7 Kit	Thermo Fisher Scientific	Cat#AM1334M
Select-a-Size^™ DNA MagBead Kit	Zymo Research	Cat#D4084
Deposited data
Mendeley Data: Raw high-throughput sequencing data for determining transcription fidelity by E. coli RNAP	This paper	dx.doi.org/10.17632/ ysc6r3dz2m.1
Recombinant DNA
pBSM	Addgene	67505
pET His6 TEV LIC cloning vector (1B)	Addgene	29653
pET LIC cloning vector (2A-T)	Addgene	29655
pET His6 LIC cloning vector (2B-T)	Addgene	29666
pET His6 LIC cloning vector (2Bc-T)	Addgene	37236
TEV protease, S219V mutant	Addgene	pRK793
Evolved sortase (eSrtA), P94R/D160N/D165A/K190E/K196T. ${His}_{6}$ at the C-terminus of eSrtA.	Chen et al., 2011¹³¹	pET29-eSrtA
Wild type E. coli RNAP with RRAS (PKA tag) + LPETG (sortag) + ${His}_{6}$ tag at the C-terminus of $β^{'}$ subunit	This paper	pIA1234
Elongation factor G with ${His}_{6}$ tag at the N-terminus	This paper	pCK-EF-G
Elongation factor Tu with ${His}_{6}$ tag at the N-terminus	This paper	pCK-EF-Tu
Elongation factor Ts with ${His}_{6}$ tag at the N-terminus	This paper	pCK-EF-Ts
Initiation factor 1 with ${His}_{6}$ tag at the C-terminus	This paper	pET24b-IF1
Initiation factor 2 with ${His}_{6}$ tag at the C-terminus	This paper	pET24b-IF2
Initiation factor 3 with ${His}_{6}$ tag at the C-terminus	This paper	pET24b-IF3
Methionyl-tRNA formyltransferase with ${His}_{6}$ tag at the C-terminus	This paper	pET2Bc-T-fmt
Nucleotide diphosphate kinase with ${His}_{6}$ tag at the N-terminus	This paper	pET2B-T-ndk
GreA with ${His}_{6}$ tag at the C-terminus	This paper	pET2Bc-T-GreA
GreB with ${His}_{6}$ tag at the C-terminus	This paper	pET2Bc-T-GreB
RelE with ${His}_{6}$ tag at the N-terminus	This paper	pET22b- $Δ9$ -His6xRelB:RelE
Alanyl-tRNA synthetase with ${His}_{6}$ tag at the N-terminus	This paper	pET2B-T-alaS
Arginyl-tRNA synthetase with ${His}_{6}$ tag at the N-terminus	This paper	pET2B-T-argS
Asparaginyl-tRNA synthetase with ${His}_{6}$ tag at the N-terminus	This paper	pET2B-T-asnS
Aspartate-tRNA synthetase with ${His}_{6}$ tag at the C-terminus	This paper	pJL-H6-aspS
Cysteinyl-tRNA synthetase with ${His}_{6}$ tag at the C-terminus	This paper	pJL-H6-cysS
Glutaminyl-tRNA synthetase with ${His}_{6}$ tag at the C-terminus	This paper	pJL-H6-glnS
Glutamyl-tRNA synthetase with His6 tag at the C-terminus	This paper	pJL-H6-gltX
Glycyl-tRNA synthetase with ${His}_{6}$ tag at the C-terminus of $β$ subunit	This paper	pJL-H6-glyQS
Histidyl-tRNA synthetase with ${His}_{6}$ tag at the C-terminus	This paper	pJL-H6-hisS
Isoleucyl-tRNA synthetase with ${His}_{6}$ tag at the N-terminus	This paper	pET2B-T-ileS
Leucyl-tRNA synthetase with ${His}_{6}$ tag at the C-terminus	This paper	pJL-H6-leuS
Lysyl-tRNA synthetase with ${His}_{6}$ tag at the C-terminus	This paper	pJL-H6-lysS
Methionyl-tRNA synthetase with ${His}_{6}$ tag at the C-terminus	This paper	pJL-H6-metG
Phenylalanyl-tRNA synthetase with ${His}_{6}$ tag at the N-terminus of the $α$ subunit	This paper	pET2B-T-pheS
Prolyl-tRNA synthetase with ${His}_{6}$ tag at the C-terminus	This paper	pJL-H6-proS
Seryl-tRNA synthetase with ${His}_{6}$ tag at the C-terminus	This paper	pJL-H6-serS
Threonyl-tRNA synthetase with ${His}_{6}$ tag at the N-terminus	This paper	pET2B-T-thrS
Tryptophanyl-tRNA synthetase with ${His}_{6}$ tag at the C-terminus	This paper	pJL-H6-trpS
Tyrosyl-tRNA synthetase with ${His}_{6}$ tag at the C-terminus	This paper	pJL-H6-tyrS
Valyl-tRNA synthetase with ${His}_{6}$ tag at the C-terminus	This paper	pJL-H6-valS
pyrL leader sequence with downstream λ phage sequence	This paper	pUC19-pyrL-TC4
tRNA^fMet isoform 1 with lpp promoter inserted into pBSM between XhoI and PstI sites	This paper	pBSM-lpp-tRNA^fMet
Pyranose oxidase from Trametes multicolor with ${His}_{6}$ tag at the C-terminus	This paper	p-PO
Software and algorithms
Igor Pro 7	WaveMetrics	https://www.wavemetrics.com/downloads/current
R	RStudio	https://www.rstudio.com
ImageQuant TL 8.2	Cytiva	https://www.cytivalifesciences.com/en/us/shop/protein-analysis/molecular-imaging-for-proteins/imaging-software/imagequant-tl-8-2-imageanalysis-software-p09518
Jupyter Notebook	Anaconda Navigator	https://docs.anaconda.com/anaconda/navigator/
Microsoft Office	Microsoft	https://www.microsoft.com/enus/microsoft-365/microsoft-office
Adobe Photoshop and Illustrator	Adobe	https://www.adobe.com
Matlab	MathWorks	https://www.mathworks.com/products/matlab.html
deML	Renaud et al., 2015¹⁶⁰	https://github.com/grenaud/deML
HTStream	Petersen et al., 2015¹⁸⁹	https://github.com/s4hts/HTStream
Calib	Orabi et al., 2019¹⁶¹	https://github.com/vpc-ccg/calib
Mafft	Katoh and Standley, 2013¹⁶⁵	https://mafft.cbrc.jp/alignment/software/
Biopython 1.19.4	Cock et al., 2009¹⁹⁰	https://biopython.org
Logomaker	Tareen and Kinney, 2020¹⁹¹	https://logomaker.readthedocs.io/en/latest/examples.html#splice-site-probability-logo
COOT v0.8.3	Emsley and Cowtan, 2004¹⁹²	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
cryoSPARC v3.1	Punjani et al., 2017¹⁷⁰	https://cryosparc.co m/
Gctf	Zhang, 2016¹⁷³	https://www2.mrc-lmb.cam.ac.uk/research/locally-developed-software/zhang-software/#gctf
MotionCor2	Zheng et al., 2017¹⁷²	https://emcore.ucsf.edu/ucsf-software
PHENIX	Adams et al., 2010¹⁹³	https://www.phenix-online.org/documentation/
RELION v3.1	Zivanov et al., 2018¹⁶⁹	https://www2.mrclmb.cam.ac.uk/relion
UCSF Chimera v 1.13.1	Pettersen et al., 2004¹⁷⁴	https://www.cgl.ucsf.edu/chimera
UCSF ChimeraX v1.0	Goddard et al., 2018¹⁹⁴	https://www.cgl.ucsfedu/chimerax/
PyMOL v1.6	Schrödinger and DeLano, 2020¹⁹⁵	https://pymol.org/2/
LocScale	Jakobi et al., 2017¹⁷⁷	https://git.embl.de/jakobi/LocScale
Single-molecule and high throughput sequencing analysis pipeline	This paper	https://zenodo.org/record/6534021#.YntNty8Rrxg
Other
10% carboxyl polystyrene 1.0 μm beads (w/w)	Bangs Laboratories, Inc.	Cat#PC04001
1260 Infinity HPLC system	Agilent	https://www.agilent.com/en/product/liquid-chromatography/hplc-systems/analytical-hplc-systems
Amicon Ultra-0.5 Centrifugal Filter Unit, 10K MWCO	Thermo Fisher Scientific	Cat#UFC501024
Amicon Ultra-0.5 Centrifugal Filter Unit, 3K MWCO	Thermo Fisher Scientific	Cat#UFC500324
Amicon Ultra-15 Centrifugal Filter Unit, 10K MWCO	Thermo Fisher Scientific	Cat#UFC901024
Amicon Ultra-15 Centrifugal Filter Unit, 30K MWCO	Thermo Fisher Scientific	Cat#UFC903008
Amicon Ultra-15 Centrifugal Filter Unit, 3K MWCO	Thermo Fisher Scientific	Cat#UFC900308
Avanti JXN-26 centrifuge	Beckman Coulter Life Sciences	Cat#
C-flat CF-1.2/1.3 400 mesh copper grids	Protochips, Inc	Cat#CF-1.2/1.3-4CU-50
Dynabeads^® M-280 Streptavidin	Thermo Fisher Scientific	Cat#11205D
Dynabeads^™ Protein G	Thermo Fisher Scientific	Cat#10003D
Econospin	Epoch Life Science	Cat#1920-050/250
EmulsiFlex-C5	Avestin	https://www.avestin.com/emulsiflexc5.htm
HiPrep^™ Sephacryl S100 16/60	GE Healthcare	Cat#17-1165-01
HiPrep^™ Sephacryl S300 16/60	GE Healthcare	Cat#17-1167-01
HisTrap HP^™	GE Healthcare	Cat#17-5248-02
HiTrap^® DEAE Fast Flow	GE Healthcare	Cat#17-5055-01
HiTrap^® Desalting	GE Healthcare	Cat#45-000-252
HiTrap^® Heparin HP	GE Healthcare	Cat#17-0407-01
HiTrap^® Q HP	GE Healthcare	Cat#17-1154-01
HiTrap^® SP HP	GE Healthcare	Cat#17-1152-01
JA-20 Fixed-Angle Aluminum Rotor	Beckman Coulter Life Sciences	Cat#334831
Janelia Fluor^® 549, Maleimide	Tocris	Cat#6500
J-LITE JLA-8.1000 Fixed-Angle Aluminum Rotor	Beckman Coulter Life Sciences	Cat#363688
Millex-GV Filter (0.22 μm)	Millipore	Cat#SLGV004SL
Mono Q^® 5/50 GL column	GE Healthcare	Cat#GE17-5166-01
Nalgene^™ Oak Ridge	Thermo Fisher Scientific	Cat#3119-0050
Ni-NTA Agarose	Qiagen	Cat#30210
Oligo Clean & Concentrator	Zymo Research	Cat#D4060
Open-Top Thick wall Polycarbonate	Beckman Coulter Life Sciences	Cat#355631
Polycarbonate Bottle with cap assembly	Beckman Coulter Life Sciences	Cat#355655 (bottle) Cat#355623 (cap)
Polypropylene Bottle Assembly	Beckman Coulter Life Sciences	Cat#A98813
S3000 Ultrasonic Liquid Processor	Misonix	Cat#EW-04711-81
SpectraPor^® dialysis membrane	Repligen	https://www.repligen.com/technologies/dialysis/spectrapor-biotech-grade-dialysis-tubing-and-membranes/spectrapor-1-5-dry-standard-grade-regenerated-cellulose-rc-dialysis-tubing-trial-kits
Steriflip	Thermo Fisher Scientific	Cat#SCGP00525
Streptavidin Magnetic Beads	Thermo Fisher Scientific	Cat#88816
SW 32 Ti Rotor	Beckman Coulter Life Sciences	Cat#369650
Syringe filter, PVDF, 0.22 μm	Genesee Scientific	Cat#25-243
Type 45 Ti Rotor	Beckman Coulter Life Sciences	Cat#339160
UltraPure^™ DNase/RNase-Free Distilled Water	Thermo Fisher Scientific	Cat#10977-023
XBridge BEH C18 Column, 130Å pore size, 5 μm particle size, 4.6 mm (inner diameter) X 150 mm (length)	Waters^™	Cat#186003116