Plasmids, DNAs and RNAs
A DNA fragment encoding B. subtilis HelD was PCR-amplified from strain MH5636 (Extended Data Table 1). The PCR product was inserted into expression vector pGEX-6p-1 via BamHI and XhoI restriction sites, in frame with a region encoding an N-terminal GST-tag. DNA fragments encoding B. subtilis σA, δ or δNTD were PCR-amplified from strain MH5636 and inserted into pETM-11 vector (EMBL, Heidelberg) via NcoI/HindIII or NcoI/XhoI restriction sites, respectively, in frame with a region encoding an N-terminal His6-tag. DNA and RNA oligomers used for the assembly of transcription complexes were purchased as single-stranded oligonucleotides (Eurofines and IBA Lifesciences, respectively).
Protein production and purification
B. subtilis strains MH5636, LK782 (ΔhelD) or LK1032 (ΔhelDΔrpoE; Extended Data Table 1) were used to produce stationary phase RNAP, RNAPΔHelD or RNAPΔδΔHelD, respectively. Chromosomes in these strains are engineered to produce a β’ subunit with a C-terminal His10-tag. Strains were grown in TB medium at 37 °C to an OD600 of 1.0 and were then shifted to 18 °C and grown to an OD600 of about 11. All purification steps were performed at 4 °C. Cells were harvested by centrifugation, resuspended in buffer A (50 mM Na2HPO4, 300 mM NaCl, 3 mM 2-mercaptoethanol, 5 % [v/v] glycerol, pH 7.9) and lysed by sonication. The lysate was cleared by centrifugation. RNAP variants were captured on Ni2+-NTA affinity resin (Macherey-Nagel), washed with buffer A supplemented with 25 mM imidazole and eluted with buffer A supplemented with 250 mM imidazole. The eluate was dialyzed overnight against 50 mM Na2HPO4, 300 mM NaCl, 3 mM DTT, 5 % [v/v] glycerol, pH 7.9, loaded on a 5 ml HiTrap Heparin HP column (GE Healthcare), washed with buffer B (50 mM TRIS-HCl, 100 mM NaCl, 3 mM DTT, 0.1 mM EDTA, 5 % [v/v] glycerol, pH 7.9) and eluted with a linear gradient to buffer B with 700 mM NaCl. Fractions containing RNAPs were pooled and further purified by SEC on a HiLoad Superdex 200 Increase 16/600 column (GE Healthcare) in 20 mM TRIS-HCl, 150 mM NaCl, 0.5 mM DTT, 5 % (v/v) glycerol, pH 8.0. The final samples were concentrated to approximately 16 mg/ml. RNAP produced from strain MH5636 was directly used for EM sample preparation. Other RNAP preparations were aliquoted, flash frozen in liquid N2 and stored at -80 °C
Recombinant B. subtilis GST-HelD was produced in Escherichia coli Rosetta(DE3) cells, His6-δ, His6-δNTD and His6-σA were produced in Escherichia coli BL21(DE3)-RIL cells. Cells were grown in auto-inducing media51 at 37 °C to an OD600 of 1.0 and further incubated at 20 °C overnight. All purification steps were performed at 4 °C. GST-HelD cells were harvested by centrifugation, resuspended in buffer C (50 mM TRIS-HCl, 500 mM NaCl, 1 mM 2-mercaptoethanol, 10 % [v/v] glycerol, pH 7.9) and lysed by sonication. The lysate was cleared by centrifugation, GST-HelD was captured on glutathione resin (Macherey-Nagel), washed with buffer C and eluted with 50 mM TRIS-HCl, 300 mM NaCl, 1 mM DTT, 10 % (v/v) glycerol, 20 mM reduced glutathione, pH 7.9. Eluted fractions were dialyzed against buffer D (20 mM TRIS-HCl, 200 mM NaCl, 1 mM DTT, 5 % [v/v] glycerol, pH 7.9) in the presence of GST-tagged PreScission protease. HelD was separated from uncleaved protein, GST and GST-PreScission by a second passage through glutathione resin. The flowthrough was further purified by SEC on a HiLoad Superdex 200 Increase 16/600 column equilibrated in buffer D. Fractions containing HelD were concentrated to approximately 15 mg/ml, aliquoted, flash frozen in liquid N2 and stored at -80 °C.
His6-δ or His6-δNTD cells were harvested by centrifugation, resuspended in 50 mM TRIS-HCl, 500 mM NaCl, 0.5 mM 2-mercaptoethanol 5 % [v/v] glycerol, pH 6.0, and lysed by sonication. The lysate was cleared by centrifugation, His6-δ/His6-δNTD was captured on Ni2+-NTA resin, washed with 50 mM TRIS-HCl, 300 mM NaCl, 0.5 mM 2-mercaptoethanol, 10 mM imidazole, 5 % (v/v) glycerol, pH 6.0, and eluted with 20 mM TRIS-HCl, 150 mM NaCl, 0.5 mM 2-mercaptoethanol, 400 mM imidazole, 5 % (v/v) glycerol, pH 6.0. For the assembly of complexes for cryoEM analysis, eluted His6-δ was supplemented with His-tagged TEV protease (1:40 [w/w]), dialyzed against buffer E (20 mM TRIS-HCl, 150 mM NaCl, 1 mM DTT, 5 % (v/v) glycerol, pH 6.0) overnight and passed through fresh Ni2+-NTA resin to remove uncleaved His6-δ, cleaved His6-tag His-tagged TEV protease. Proteins were further purified by SEC on a Superdex75 Increase 10/300 column (GE Healthcare) in buffer E. Fractions containing His6-δ, δ or His6-δNTD were concentrated to approximately 4 mg/ml (His6-δ, His6-δNTD) and 23 mg/ml (δ), aliquoted, flash frozen in liquid N2 and stored at -80 °C.
σA cells were harvested by centrifugation, resuspended in buffer F (20 mM TRIS-HCl, 500 mM NaCl, 1 mM 2-mercaptoethanol, 5 % [v/v] glycerol, pH 7.5) supplemented with 20 mM imidazole, and lysed by sonication. The lysate was cleared by centrifugation, His6-σA was captured on Ni2+-NTA resin, washed with buffer F supplemented with 50 mM imidazole, and eluted with buffer F supplemented with 400 mM imidazole. Eluted His6-σA was supplemented with His-tagged TEV protease (1:40 [w/w]), dialyzed against buffer F supplemented with 1 mM EDTA overnight and passed through fresh Ni2+-NTA resin to remove uncleaved His6-σA, cleaved His6-tag and His-tagged TEV protease. The target protein was further purified by SEC on a Superdex75 Increase 16/600 column (GE Healthcare) in 25 mM TRIS-HCl, 300 mM NaCl, 0.1 mM DTT, 5 % (v/v) glycerol, pH 7.5. Fractions containing σA were concentrated to approximately 39 mg/ml, aliquoted, flash frozen in liquid N2 and stored at -80 °C.
Crosslinking/mass spectrometry
Sulfo-SDA predominantly establishes lysine-X crosslinks through a primary amine-reactive moiety on one side and a UV-activatable moiety on the other (theoretical crosslinking limit 25 Å). Sulfo-SDA was prepared at 3 mg/ml in 20 mM HEPES-NaOH, 5 mM Mg(OAc)2, 300 mM NaCl, 5 mM DTT, 5% (v/v) glycerol, pH 8.0 immediately prior to addition of RNAPΔδΔHelD, RNAPΔδΔHelD-δ, RNAPΔδΔHelD-HelD or RNAPΔδΔHelD-δ-HelD (protein:sulfo-SDA 1:3 [w/w]). Samples were incubated on ice for two hours and then irradiated in a thin film using 365 nm UV irradiation (UVP CL‐1000 UV Crosslinker, UVP Inc.) for 20 min on ice (5 cm distance from UV-A lamp). The crosslinked samples were separated by 4-12 % BIS-TRIS NuPAGE, gel bands corresponding to crosslinked monomeric complexes were excised and digested in-gel as described previously52 Resulting peptides were desalted using C18 StageTips53.
10 % of each sample were analyzed by LC-MS/MS without fractionation, the remaining 90 % were fractionated using SEC on a Superdex Peptide 3.2/300 column (GE Healthcare) in 30 % (v/v) acetonitrile, 0.1 % (v/v) trifluoroacetic acid at a flow rate of 10 µl/min to enrich for crosslinked peptides54. The first six peptide-containing fractions (50 μl each) were collected, solvent was removed using a vacuum concentrator and the fractions were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific), connected to an Ultimate 3000 RSLCnano system (Dionex, Thermo Fisher Scientific).
The non-fractionated samples were injected onto a 50 cm EASY-Spray C18 LC column (Thermo Fisher Scientific) operated at 50 °C. Peptides were separated using a linear gradient going from 2 % mobile phase B (80 % [v/v] acetonitrile, 0.1 % [v/v] formic acid) to 40 % mobile phase B in mobile phase A (0.1 % [v/v] formic acid) at a flow rate of 0.3 μl/min over 110 minutes, followed by a linear increase from 40 % to 95 % mobile phase B in 11 minutes. Eluted peptides were ionized by an EASY-Spray source (Thermo Fisher Scientific) and MS data were acquired in the data-dependent mode with the top-speed option. For each three-second acquisition cycle, the full scan mass spectrum was recorded in the Orbitrap with a resolution of 120,000. The ions with a charge state from 3+ to 7+ were isolated and fragmented using higher-energy collisional dissociation (HCD) with 30 % collision energy. The fragmentation spectra were then recorded in the Orbitrap with a resolution of 50,000. Dynamic exclusion was enabled with single repeat count and 60 s exclusion duration.
SEC fractions were analyzed using an identical LC-MS/MS setup. Peptides were separated by applying a gradient ranging from 2 % to 45 % mobile phase B (optimized for each fraction) over 90 min, followed by ramping up mobile phase B to 55 % and 95 % within 2.5 min each. For each three-second data-dependent MS acquisition cycle, the full scan mass spectrum was recorded in the Orbitrap with a resolution of 120,000. The ions with a charge state from 3+ to 7+ were isolated and fragmented using HCD. For each isolated precursor, one of three collision energy settings (26 %, 28 % or 30 %) was selected for fragmentation using a data-dependent decision tree based on the m/z and charge of the precursor. The fragmentation spectra were recorded in the Orbitrap with a resolution of 50,000. Dynamic exclusion was enabled with single repeat count and 60 s exclusion duration.
LC-MS/MS data generated from the four complexes were processed separately. MS2 peak lists were generated from the raw MS data files using the MSConvert module in ProteoWizard (version 3.0.11729). The default parameters were applied, except that Top MS/MS Peaks per 100 Da was set to 20 and the denoising function was enabled. Precursor and fragment m/z values were recalibrated. Identification of crosslinked peptides was carried out using xiSEARCH software (https://www.rappsilberlab.org/software/xisearch; version 1.7.4)55. For RNAPΔδΔHelD, peak lists were searched against the sequence and the reversed sequence of RNAP subunits (α, β, β’ and ε) and two co-purified proteins, σA and σB. For RNAPΔδΔHelD-δ, RNAPΔδΔHelD-HelD and RNAPΔδΔHelD-δ-HelD samples, protein sequences of δ, HelD or both were included in the database. The following parameters were applied for the search: MS accuracy = 4 ppm; MS2 accuracy = 8 ppm; enzyme = trypsin (with full tryptic specificity); allowed number of missed cleavages = 2; missing monoisotopic peak = 2; crosslinker = sulfo-SDA (the reaction specificity for sulfo-SDA was assumed to be for lysine, serine, threonine, tyrosine and protein N termini on the NHS ester end, and any amino acid residue for the diazirine end); fixed modifications = carbamidomethylation on cysteine; variable modifications = oxidation on methionine and sulfo-SDA loop link. Identified crosslinked peptide candidates were filtered using xiFDR56. A false discovery rate of 5 % on residue-pair level was applied with the “boost between” option selected. Crosslinked residue pairs identified from the four complexes are summarized in Extended Data Table 4 and Supplementary Table 1.
Cryo-EM data collection and processing
Equimolar amounts of tDNA, ntDNA and RNA were mixed in buffer G (20 mM TRIS-HOAc, 5 mM Mg[OAc]2, 100 mM KOAc, 2 mM DTT, 5 % [v/v] glycerol, pH 8.0) and annealed by heating to 95 °C for 5 min and subsequent cooling to 25 °C at 1 °C/min. The annealed scaffold was incubated with B. subtilis RNAP in a 1.3:1 molar ratio in buffer H (20 mM TRIS-HOAc, 5 mM Mg[OAc]2, 300 mM KOAc, 2 mM DTT, 5 % [v/v] glycerol, pH 8.0) for 10 min on ice, then for 10 min at 32 °C. Equimolar amounts (to RNAP) of δ and HelD were added stepwise, followed by incubation for 10 min at 32 °C after each addition. The mixture was subjected to SEC on a Superdex 200 Increase 3.2/300 column (GE Healthcare) in buffer H. Fractions containing RNAP, δ and HelD were pooled and concentrated to approximately 5 mg/ml.
Immediately before preparation of the grids, the sample was supplemented with 0.15 % (w/v) n-octylglucoside. 3.8 µl of the final mixture were spotted on plasma-treated Quantifoil R1/2 holey carbon grids at 10 °C/100 % humidity, and plunged into liquid ethane using a FEI Vitrobot Mark IV. Image acquisition was conducted on a FEI Titan Krios G3i (300 kV) with a Falcon 3EC camera at a nominal magnification of 92,000 in counting mode using EPU software (Thermo Fisher Scientific) with a calibrated pixel size of 0.832 Å. A total electron dose of 40 e/Å2 was accumulated over an exposure time of 36 s. Movie alignment was done with MotionCor257 using 5x5 patches followed by ctf estimation with Gctf58.
All following image analysis steps were done with cryoSPARC59. Class averages of manually selected particles were used to generate an initial template for reference-based particle picking from 9,127 micrographs. Particle images were extracted with a box size of 440 and binned to 110 for initial analysis. Ab initio reconstruction using a small subset of particles was conducted to generate an initial 3D reference for 3D heterogeneous refinement. The dataset was iteratively classified into two well-resolved populations representing monomeric and dimeric RNAP-δ-HelD. Selected particles were re-extracted with a box of 220 and again classified in 3D to further clean the dataset. Finally, selected particle images were re-extracted with a box of 280 (1.3 Å/px) and subjected to local refinement using a generously enlarged soft-mask for monomeric or dimeric RNAP-δ-HelD. Local refinement of the dimer particles using the monomeric mask was conducted as a control to trace differences of RNAP-δ-HelD in the authentic monomer and dimer structures. After per-particle CTF correction, non-uniform refinement was applied to generate the final reconstructions.
Model building and refinement
The final cryoEM map for the dimeric RNAP-δ-HelD complex (Extended Data Fig. 3) was used for initial model building. Coordinates of M. smegmatis RNAP α, β and β’ subunits (PDB ID 5VI8)60 were docked into the cryoEM map using Coot61. Modeling of δ was based on the NMR structure of B. subtilis δ (PDB ID 2M4K)62. Modeling of ε was supported by the structure of YkzG from Geobacillus stearothermophilus (PDB ID 4NJC)19. Model building of HelD was supported by the structure of UvrD helicase from E. coli (PDB ID 3LFU)63 as well as the C-terminal domain of a putative DNA helicase from Lactobacillus plantarun (PDB ID 3DMN). The subunits were manually rebuilt into the cryoEM map. The model was completed and manually adjusted residue-by-residue, supported by real space refinement in Coot. The manually built model was refined against the cryoEM map using the real space refinement protocol in PHENIX64. Model building of the monomeric complex was done in the same way but starting with a model of half of the dimeric complex. The structures were evaluated with Molprobity65. Structure figures were prepared using PyMOL (Version 1.8 Schrödinger, LLC).
Structure comparisons
Structures were compared by global superposition of complex structures or by superposition of selected subunits in complexes using the “secondary structure matching” algorithm implemented in Coot or the “align” algorithm implemented in PyMOL.
Size exclusion chromatography/multi-angle light scattering
SEC/MALS analysis was performed on an HPLC system (Agilent) coupled to mini DAWN TREOS multi-angle light scattering and RefractoMax 520 refractive index detectors (Wyatt Technology). RNAP-δ-HelD complex was assembled as for cryoEM. 60 μl of the sample at 1 mg/ml were chromatographed on a Superose 6 Increase 10/300 column (GE Healthcare) in buffer H supplemented with 0.02 % (w/v) NaN3 at 18 °C with a flowrate of 0.6 ml/min. Data were analyzed with the ASTRA 6.1 software (Wyatt Technology) using monomeric bovine serum albumin (Sigma-Aldrich) as a reference.
Interaction assays
HelD interactions with δ or δNTD were analyzed by analytical SEC. 21 µM HelD and 42 µM δ or δNTD were mixed in 20 mM HEPES-NaOH, 50 mM NaCl, 1 mM DTT, pH 7.5, and incubated for 10 min at room temperature. 50 µl of the samples were loaded on a Superdex S200 Increase PC 3.2 column (GE Healthcare) and chromatographed at 4 °C with a flow rate of 40 µl/min. Fractions were analyzed by 12.5 % SDS PAGE.
Electrophoretic gel mobility shift assays
Equimolar amounts of 5’-[32P]-labeled tDNA and unlabeled ntDNA were mixed in buffer G and annealed by heating to 95 °C for 5 min and subsequent cooling to 25 °C at 1 °C/min. 1 µM each of HelD, δ, RNAPΔδΔHelD, RNAPΔδΔHelD/HelD, RNAPΔδΔHelD/δ or 1 µM RNAPΔδΔHelD/HelD and increasing amounts of δ or δNTD were mixed with 5 nM of the labeled duplex, and incubated in buffer H for 10 min at 4 °C followed by 10 min at 32 °C. Samples were loaded on a 4 % native PAGE gel and electrophoresed in 0.5X TBE buffer. Radioactive bands were visualized using a Storm phospohorimager and quantified using ImageQuant software (GE Healthcare).
HelD release assays
Equimolar amounts of HelD and stationary phase RNAP were mixed in buffer I (20 mM TRIS-HCl, 300 mM NaCl, 2 mM DTT, 5 % (v/v) glycerol, pH 8.0), incubated for 10 min on ice and then for 10 min at 32 °C. The sample was chromatographed on a HiLoad Superdex 200 Increase 10/300 column (GE Healthcare) in buffer I. Fractions were analyzed by 12.5 % SDS PAGE, fractions containing RNAP-HelD complex were collected and concentrated to approximately 3 mg/ml (6.7 µM). 80 µl 6.7 µM complex were mixed with buffer I, 5 mM Mg2+-ATPγS/AMPPNP/ATP/ADP/AMP, 6.7 µM σA or σA plus Mg2+-ATPγS in buffer I. 90 µl of the samples were loaded on a Superdex S200 Increase PC 3.2 column (GE Healthcare) and chromatographed at 4 °C with a flow rate of 40 µl/min. Fractions were analyzed by 12.5 % SDS PAGE.