==== Front Nat Commun Nat Commun Nature Communications 2041-1723 Nature Publishing Group UK London 20134 10.1038/s41467-020-20134-y Article The bacterial multidrug resistance regulator BmrR distorts promoter DNA to activate transcription http://orcid.org/0000-0002-8483-7902Fang Chengli 12 Li Linyu 3 Zhao Yihan 4 Wu Xiaoxian 12 Philips Steven J. 5 You Linlin 12 Zhong Mingkang 3 Shi Xiaojin 3 http://orcid.org/0000-0001-8732-5059O’Halloran Thomas V. 567 Li Qunyi liqunyi@huashan.org.cn 3 http://orcid.org/0000-0002-1778-8389Zhang Yu yzhang@sippe.ac.cn 1 1 grid.9227.e0000000119573309Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, 200032 Shanghai, China 2 grid.410726.60000 0004 1797 8419University of Chinese Academy of Sciences, 100049 Beijing, China 3 grid.8547.e0000 0001 0125 2443Clinical Pharmacy Laboratory, Huashan Hospital, Fudan University, 200040 Shanghai, China 4 grid.256922.80000 0000 9139 560XKey Laboratory of Plant Stress Biology, State Key Laboratory of Cotton Biology, School of Life Sciences, Henan University, 475004 Kaifeng, China 5 grid.16753.360000 0001 2299 3507Department of Chemistry, Northwestern University, Evanston, IL 60208 USA 6 grid.16753.360000 0001 2299 3507Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208 USA 7 grid.16753.360000 0001 2299 3507The Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208 USA 8 12 2020 8 12 2020 2020 11 628420 7 2020 10 11 2020 © The Author(s) 2020Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.The MerR-family proteins represent a unique family of bacteria transcription factors (TFs), which activate transcription in a manner distinct from canonical ones. Here, we report a cryo-EM structure of a B. subtilis transcription activation complex comprising B. subtilis six-subunit (2αββ‘ωε) RNA Polymerase (RNAP) core enzyme, σA, a promoter DNA, and the ligand-bound B. subtilis BmrR, a prototype of MerR-family TFs. The structure reveals that RNAP and BmrR recognize the upstream promoter DNA from opposite faces and induce four significant kinks from the −35 element to the −10 element of the promoter DNA in a cooperative manner, which restores otherwise inactive promoter activity by shortening the length of promoter non-optimal −35/−10 spacer. Our structure supports a DNA-distortion and RNAP-non-contact paradigm of transcriptional activation by MerR TFs. BmrR is a member of the bacterial MerR transcription factor family that regulates the expression of the bacterial efflux pump Bmr. Here, the authors present the cryo-EM structure of a B. subtilis transcription activation complex (TAC) containing the RNA Polymerase (RNAP) core enzyme, σA, promoter DNA and ligand-bound BmrR, which reveals that in contrast to most other transcription factors BmrR does not directly interact with RNAP and instead activates transcription by modulating the shape of the core promoter. Subject terms BiochemistryBacterial transcriptionCryoelectron microscopyhttps://doi.org/10.13039/501100002855Ministry of Science and Technology of the People’s Republic of China (Chinese Ministry of Science and Technology)2018YFA0900701Zhang Yu https://doi.org/10.13039/501100002367Chinese Academy of Sciences (CAS)XDB29020000QYZDB-SSW-SMC005Zhang Yu https://doi.org/10.13039/501100001809National Natural Science Foundation of China (National Science Foundation of China)3182200131970040Zhang Yu issue-copyright-statement© The Author(s) 2020 ==== Body Introduction Transcription activation involves cooperative interplay among RNA polymerase (RNAP) holoenzyme, promoter DNA, and transcription factors (TFs)1,2. The prevailing “recruitment” model of bacterial transcription activation specifies that TFs enrich RNAP at specific genomic loci by making direct interactions with both RNAP and promoter DNA, thereby creating additional linkage between them3. In line with their function, canonical bacterial TFs are typically equipped with a DNA-binding domain (DBD), which reads specific sequence in the form of double-stranded DNA (dsDNA), and small surface patches, which interact with the RNAP-α subunit or domain R4 of σ factors (σ4)4,5. Previous biochemical and structural evidence suggests that the MerR-family TFs probably activate transcription in a manner distinct from the canonical TFs6–12. The MerR-family TFs, based on protein domain architecture, are divided into two subgroups. The first subgroup is composed of metal- or oxidative stress-responsive factors, which are ultrasensitive to cellular metal ions, such as Escherichia coli CueR13, E. coli ZntR14, Ralstonia metallidurans PbrR15, Pseudomonas putida CadR16, and Synchocystis PCC 6803 CoaR17, or to cellular superoxide, such as E. coli SoxR18. The second subgroup is composed of multidrug-responsive regulators, which are capable of binding multiple compounds with diverse chemical properties, such as Bacillus subtilis BmrR19, B. subtilis BltR20, and B. subtilis Mta21. MerR-TFs recognize a long palindromic operator that locates in a region completely overlapping with the spacer between the −35 and −10 elements (two key regions of promoter DNA for RNAP recognition) of their regulated promoter DNA1,6,8,21. The reported crystal structures of binary complexes of MerR-family TFs bound with their cognate operator DNA revealed a striking distortion around the center of their palindromic operators7,8,12,18. Given that the MerR-TF-regulated promoters contain an unusually long 19-/20-bp spacer compared with the regular promoters with an optimal 17-bp spacer between the −35 and −10 elements1,6,8,21, such DNA distortion was proposed to realign the geometry of the non-optimal promoter DNA and to restore the promoter activity7,9,22. However, due to the lack of RNAP in those crystal structures, it is unknown how MerR-TFs manage to reconcile with RNAP to bind and reshape promoter DNA with near-complete spatial overlapping binding site for RNAP (spanning from −40 to +20) and MerR-family TFs (spanning from −35 to −15). Bacillus subtilis BmrR is the prototype of the second subgroup of MerR-TFs7,23–25. It regulates the expression of Bmr (bacterial multidrug resistance), a bacterial efflux pump recognizing cationic amphiphilic chemicals19. Similar to the substrate spectrum of the Bmr efflux pump, BmrR is capable of recognizing xenobiotic cationic chemicals with a broad range of structural, chemical, and binding properties24,26–28. A large collection of the reported crystal structure of BmrR apoprotein and BmrR complexed with drug and DNA reveal substantial conformational dynamics of BmrR7,23–25,29,30, thereby providing a foundation to explore the conformation change upon RNAP recruitment and the underlying transcription activation mechanism of MerR-TFs. In the present study, we report a cryo-EM structure of transcription activation complex (TAC) with B. subtilis BmrR at 4.4 Å. The structure shows that RNAP and BmrR bind at the opposite faces of the promoter DNA. Albeit barely interacting with each other, RNAP and BmrR together induce four kinks at the upstream promoter dsDNA, which shortens the non-optimal −35/−10 spacer for efficient promoter unwinding. Our work supports an RNAP-non-contact transcription activation paradigm only involving DNA distortion. Results The overall structure of BmrR-TAC To assemble the BmrR-TAC, we purified endogenous B. subtilis RNAP-σA holoenzyme and recombinant B. subtilis BmrR (Supplementary Fig. 1). We confirmed that BmrR is able to activate pbmr in the presence of tetraphenylphosphonium (TPP) (Supplementary Fig. 1c). Bacterial TACs and transcription initiation complexes assembled using the pre-melted promoter DNA exhibited almost identical conformation and interactions between protein and DNA as the complexes obtained using duplex promoter DNA31–34. Therefore, to improve the sample homogeneity and enhance protein–DNA interaction, we designed a Pbmr chimeric promoter DNA comprising a 27-bp (−38 to −12) upstream promoter dsDNA of the wild-type pbmr DNA, a 13-bp (−11 to +2) non-complementary transcription bubble with consensus sequence at the −10 element, the discriminator element, and the core-recognition element, and a 10-bp (+3 to +12) downstream promoter dsDNA with G/C-rich sequences (Fig. 1a and Supplementary Fig. 1a). BmrR is capable of promoting RNAP-promoter DNA open complex (RPo) formation of both the wild-type and chimeric Pbmr promoters (Supplementary Fig. 1d).Fig. 1 The cryo-EM map and model of B. subtilis BmrR transcription activation complex (BmrR-TAC). a Nucleic acid scaffold used for cryo-EM structure determination of BmrR-TAC. Black diamond, the center of BmrR operator; orange, non-template DNA; red, template DNA; light blue, −35 element; pink, −10 element; cyan, discriminator element; blue, core-recognition element; purple, RNA; dashed box, BmrR operator DNA; gray shaded box, disordered nucleotides in the structure; red box, sequenced modified for improving complex stability; and blue shaded box, the extended −10 region. b The top and front view-orientations of cryo-EM map of B. subtilis BmrR-TAC. The RNAP subunits, nucleic acids, and BmrR are colored as in the color scheme. c The cryo-EM map and model for B. subtilis σA. d The cryo-EM map and model for B. subtilis BmrR dimer. The box highlights the map for tetraphenylphosphonium (TPP) in the BmrR-LBD. e The cryo-EM map and model for the promoter DNA. The inset box highlights the promoter central kink. f The cryo-EM map for B. subtilis RNAP-ε subunit. The cryo-EM maps were contoured at 0.00924 and structure figures were prepared in Chimera X. The catalytically competent BmrR-TAC was reconstituted using the Bs RNAP holoenzyme, TPP-bound Bs-BmrR, and the above-mentioned nucleic-acid scaffold (Supplementary Fig. 1e–h). The final cryo-electron microscopy (cryo-EM) map, reconstructed using a total of 103,226 single particles, was refined to a nominal resolution of 4.4 Å (Supplementary Fig. 2a–c), with ~4 Å at the center of RNAP and ~6 Å at the peripheral BmrR (Supplementary Fig. 2d). The cryo-EM map shows a clear signal for BmrR dimer, 50-bp promoter DNA (−38 to +12) with the exception of disordered template ssDNA (−11 to +2), five major domains of B. subtilis σA subunit (σA1.2, σA2, σA3.1, σA3.2, and σA4), and six subunits of Bs RNAP (two α subunit, one β subunit, one β′ subunit, and one ω subunit, as well as the ε subunit) (Fig. 1 and Supplementary Fig. 3a, b). In addition to the universally conserved five subunits (2αββ′ω), B. subtilis RNAP contains two additional small subunits, one of which is the RNAP-ε subunit, encoded by B. subtilis rpoY and broadly present in phylum Firmicute35. Although it was identified 40 years ago36, it is still unknown how it interacts with the rest of RNAP core enzyme. Our cryo-EM map of B. subtilis BmrR-TAC structure shows a clear signal for the RNAP-ε subunit (Fig. 1f), which locates in a cavity created by RNAP-α, -β, and -β′ subunits at the base of RNAP core enzyme, a location far away from the RNAP active-center cleft and distinct from that of its structurally related phage protein T7 GP236,37. Although no defect of transcription activity and bacteria growth was observed in the absence of RNAP-ε subunit36, the RNAP-ε subunit makes extensive interactions with the RNAP-α, -β, and -β′ subunits (Supplementary Fig. 3), suggesting that it might be involved in stability or assembly of RNAP core enzyme. Cooperative recognition of promoter DNA by BmrR and RNAP In the BmrR-TAC structure, the entire promoter DNA was protected by RNAP and BmrR dimer (Fig. 2a). The B. subtilis BmrR-TAC structure shows a closed-clamp conformation of RNAP and properly engaged transcription bubble and downstream dsDNA in the RNAP main cleft as previously reported RPo structures (Supplementary Fig. 4a–e)31,32.Fig. 2 The overall structure of BmrR-TAC. a The top and front view-orientations of the BmrR-TAC structure. b Structure superimposition of BmrR/DNA in BmrR-TAC and BmrR/DNA in the crystal structure of a puromycin-bound BmrR/DNA complex (PDB: 3Q3D). c Major (M) and minor (m) groove widths of BmrR operator dsDNA in the BmrR-TAC structure and BmrR/DNA binary complex. BmrR dimerizes and makes interactions with its operator DNA essentially as in previously reported crystal structure of BmrR–DNA complexes7,24, suggesting that RNAP engagement induces neither significant conformational change to BmrR dimer nor changes of the interaction between BmrR dimer and its operator DNA (Fig. 2b, c). Briefly, the two BmrR protomers (BmrRUS and BmrRDS) were zippered together through the central helices (residues 77–115) of two protomers and further glued by an interaction between the DBD (BmrR-DBD) of one protomer and the ligand-binding domain (BmrR-LBD) of the other protomer (Fig. 2b). The central zipper helix adopts a straight conformation, as observed in the crystal structure of BmrR–DNA complexes (Supplementary Fig. 5a)7. Although the current resolution does not permit unambiguous modeling of the small-molecular ligand TPP, the cryo-EM map shows a strong signal in the ligand-binding site identical to the location of tetraphenylantimonium (TPSb, an analog of TPP) in the crystal structure of TPSb-bound BmrR7, suggesting presence of the compound in the structure (Fig. 1d; insertion box). The BmrR-DBD of each protomer adopts a winged helix–turn–helix (wHTH) fold and contacts the major groove and minor groove using its HTH and wing loop motifs, respectively (Fig. 3a, b).Fig. 3 Cooperative recognition of promoter DNA by BmrR and RNAP. a The upstream promoter dsDNA is fully protected by BmrR and σA (surface and cartoon presentation). b BmrRUS and σA4 protects the upstream half of BmrR operator at opposite DNA faces. c BmrRDS and σA3.1 protect the downstream half of BmrR operator at opposite DNA faces. d BmrR has little contact with RNAP-σA holoenzyme. The colors are as in Fig. 1. Although the −35 element and the upstream half of BmrR operator (BmrR-OUS; −36 to −25) are partially overlapped, the first three positions (−37 to −35) of the −35 element retain consensus sequence (5′-TTG-3′) for most of the BmrR-regulated promoters (Fig. 1a and Supplementary Table 2), and σA4 recognizes the three nucleotides of the −35 element in BmrR-TAC in a manner essentially the same as it does in the crystal structure of σA4/−35 DNA38 (Supplementary Fig. 4d). The wing loop of BmrRUS-DBD inserts into the minor groove of the last three position (−34 to −32) of the −35 element and recognizes its operator sequence without interfering with the interaction between σA4 and promoter DNA (Fig. 3a, b). Such mode of interaction allows BmrRUS and σA4 recognizing their respective sequences around the −35 region from the opposite faces of promoter DNA involving no protein–protein interaction (Fig. 3b). This architecture of BmrR-TAC is in sharp contrast to the assembly of bacterial TACs with class II transcription TFs, which make extensive interaction with σA4 and RNAP-α subunit, overtake the −35 element of promoter DNA, and result in a loosed interaction of σA4 on the degenerate −35 element4. The downstream BmrR operator BmrR-ODS completely overlaps with the extended −10 region of the promoter DNA (Fig. 1a). The BmrRDS-DBD recognizes its operator DNA by placing the recognition helix and wing loop into the major and minor grooves on the same face of the promoter DNA, respectively, in a manner that allowing the σA3.1 to access the major groove of the promoter DNA from the opposite face in a sequence-independent fashion (Fig. 3c). Similar to BmrRUS, BmrRDS makes no interaction with either σA or RNAP core enzyme (Fig. 3d). In the promoter regions of downstream BmrR operator, the Bs RNAP-σA holoenzyme unwinds the −10 element and accommodates the transcription bubble and downstream dsDNA in a similar manner as other bacterial RNAP (Supplementary Fig. 4b–e)31,32,39. BmrR and RNAP induce distortion of upstream promoter DNA The upstream promoter dsDNA is significantly distorted by BmrR and σA compared with the near straight upstream dsDNA in E. coli RPo (Fig. 4a, b and Supplementary Fig. 4f). We observed a large kink (kink 3) at the center of its palindromic operator DNA (−24) accompanied by a widened minor groove and a narrowed major groove around the unpaired central two A:T base pairs (Figs. 1e and 2c)7. Notably, such kink causes a 90° sharp turn of the helical axis of the upstream promoter dsDNA (Fig. 4a), similar to the TBP-distorted TATA box in eukaryotic transcription pre-initiation complexes40. Previous reported binary crystal structures of BmrR–DNA complexes also show a similar but less kinked conformation and unpaired central A:T base pairs of its operator DNA when complexed with a ligand-bound BmrR or a constitutively active derivative of BmrR, indicating that the central kink is mainly attributed to ligand-bound BmrR binding and is further enlarged by RNAP approaching, and also suggesting that such kinked conformation of promoter DNA is required for transcription activation of BmrR (Fig. 2b, c and Supplementary Fig. 5b).Fig. 4 BmrR and RNAP induce four kinks of upstream promoter dsDNA. a The kinks of the upstream promoter DNA at positions −35 (∠①, 37°), −31 (∠②, 52°), −24 (∠③, 90°), and −18 (∠④, 48°). The colors are as in Fig. 1. b Comparison of the upstream promoter dsDNA in BmrR-TAC and E. coli RPo (PDB: 6OUL). c The superimposition of upstream promoter dsDNA in B. subtilis BmrR-TAC (cyan) and E. coli CueR-TAC (yellow). d The distribution of −35/−10 spacer length of MerR-TF-regulated promoters reported in the literature (see Supplementary Table 2 for promoter sequences). In addition to the central kink (kink 3), we observed three additional kinks at the upstream promoter dsDNA. Kink 1 corresponds to a downward 37° bend of the helical axis at the center of the −35 element (−35); kink 2 corresponds to a upward 52° bend of the helical axis at the middle point (−31) of the DNA region contacted by wing loop and HTH motifs of BmrRUS; and the kink 4 corresponds to an upward 48° bend of the helical axis at the downstream middle point (−18), the DNA region contacted by the wing loop and HTH motifs of BmrRDS (Fig. 4a). The kink 1 is imposed by σA4 and BmrRUS. The kink 2 and kink 4 are initially imposed by the BmrR dimer as observed in the crystal structure of BmrR–DNA complex, but are further enlarged by σA4 and σA3.1 during engagement of RNAP holoenzyme (Supplementary Fig. 5c–e). Altogether, we propose that ligand-bound BmrR pre-distorts its operator DNA for efficient RNAP recruitment, which further sub-tunes the promoter geometry. The net effect of the four kinks induced by RNAP and BmrR realigns the −35 and −10 regions of promoter dsDNA to the optimal interspace length and geometry to activate transcription from the otherwise silent promoter, which will be discussed in the following section. Discussion σA4 and σA2, the two major domains of σ factors, are anchored on the surface of RNAP core enzyme with little flexibility, thereby the RNAP-σA holoenzyme could only recognize promoters with a confined length of spacer between the −35 element, recognized by σA4, and the −10 element, recognized by σA2. Indeed, the majority of bacterial promoter DNAs contain the −35/−10 spacer of 17 ± 1 bp41. Recent work from Darst’s lab has trapped a near-complete set of intermediate states during RPo formation and for the first time provided a full picture of the complicated process of promoter unwinding using an rps T2 promoter with a −35/−10 spacer of 17 bp42. The structures show that RNAP-σ70 first recognizes the −35 element in a sequence-dependent manner, places the downstream straight dsDNA on top of the RNAP-β protrusion, and interacts with −10 element in a sequence-independent manner in the RPc. Subsequently, a rotation of dsDNA at the −10 element precisely places the −12/−11 base pairs near the tryptophan dyad (a key structural motif for separating promoter dsDNA at the upstream junction) and the −11A pocket (another key structural motif for recognizing and trapping base of the first unwound and flipped nucleotide), resulting in the spontaneous unwinding the −11 base pair and securing the non-template −11A into its pocket at the stage of RPi1 and RPi1.5 (Fig. 5a).Fig. 5 The proposed mechanism of transcription activation by BmrR. a The simplified pathway of rps T2 promoter (17-bp −35/−10 spacer) unwinding by E. coli RNAP-σ70 holoezyme. The Ec T-RPc (PDB: 6PSQ; top panel), RPi1.5a (PDB: 6PSS; middle panel), and RPo (PDB: 6PSW; bottom panel) were adapted from ref. 42. b The proposed aborted pathway for unwinding Pbmr by B. subtilis RNAP-σA holoenzyme. The Pbmr-RPc (upper panel) was built base on the Ec T-RPc (PDB: 6PSQ); the hypothetic intermediate of Pbmr unwinding by B. subtilis RNAP-σA promoter, in which the −12 T:A base pair and −11A is far away from the W-dyad and −11A pocket, two key structural elements for protein unwinding, and the −13T(nt) is not able to be secured by the −11A pocket. c The proposed pathway for TPP-BmrR-bound Pbmr unwinding by B. subtilis RNAP-σA holoenzyme. The Pbmr-BmrR-RPc (upper panel) was built base on the Ec T-RPc (PDB: 6PSQ); the Pbmr-BmrR-RPi1.5a (middle panel) was built base on the Ec T-RPc (PDB: 6PSQ); and the Pbmr-BmrR-RPo was determined in this study. Red, template DNA; orange, non-template DNA; blue, σA; cyan, BmrR; yellow patch, W-dyad; purple circle, the −11A pocket. In a scenario of a promoter with a non-optimal −35/−10 spacer, such as the BmrR-regulated Pbmr containing a 19-bp spacer (2-bp offset from the optimal 17-bp spacer), we infer that the RNAP-σA is able to locate and bind the −35 element, and subsequently place the straight dsDNA on top of the RNAP-β protrusion as it does for the promoter with a 17-bp spacer. However, at the stage of RPi1 formation, the rotation of promoter is not able to position the −12/−11 base pairs near the two key structural motifs, especially the spontaneously flipped −11A is far from its pocket, resulting in the failure of initiating of promoter unwinding and subsequent quick dissociation of the unstable complex (Fig. 5b). In the presence of ligand-bound BmrR, although the upstream DNA is highly distorted, the four complex kinks induced by RNAP and BmrR together realign the helical axis of the −35 and −10 regions, allowing the −35 and −10 regions of promoter dsDNA to load on σ4 (in a sequence-specific manner) and the top of RNAP-β protrusion (in a sequence-nonspecific manner), respectively, as in the modeled BmrR-RPc (Fig. 5c). More importantly, the four complex kinks shorten the distance between the −35 and −10 elements, restore the proper stereochemistry of the two elements, position the −12/−11 base pairs near the tryptophan dyad and the −11A pocket ready for the subsequent spontaneous unwinding of the −11 base pair and securing the non-template −11A into −11A pocket in a BmrR-RPi1.5 structure model (Fig. 5c), and finally leads to the formation of a stable BmrR-TAC as determined in the study. Structure superimposition of the B. subtilis BmrR-TAC and E. coli CueR-TAC (containing the other prototype member of the MerR-TF family, CueR, that also activates promoters with a 19-bp non-optimal −35/−10 spacer; determined in our parallel study) unveils the same four complex kinks at the upstream promoter dsDNA (Fig. 4c)43. On the other hand, a survey of 28 experimentally confirmed promoters regulated by known MerR-family TFs reveals that all promoters contain 19- or 20-bp spacers between the −35 and −10 elements (Fig. 4d), suggesting that the MerR-family TFs probably activate transcription by using the same DNA-distortion and RNAP-non-contact mechanism. The bacterial RNAP-σ70 holoenzyme itself could accommodate promoter DNA with 17 ± 1 bp spacer by rotating the σ4 domain anchored on the flexible tip helix of the β flap44. Thereby, BmrR probably binds and orients 20-bp spacer DNA in a similar manner as it does for 19-bp spacer DNA and the rotation of σ4 domain accommodates the additional base pair. The majority of TFs employ RNAP–protein contacts to recruit RNAP for initiating transcription of downstream genes. Given the significant overlapping binding sites of MerR-TFs and σ factor, it is proposed that transcription activation by MerR-TFs also involves RNAP interaction besides DNA distortion45. This study resolves the long-standing question by showing that the MerR-TFs activate transcription solely through DNA distortion involving no interaction with RNAP holoenzyme, and thus support an uncanonical paradigm of RNAP-non-contact transcription activation. This transcription regulation mechanism provides a precedent for a variety of other eukaryotic factors that work by manipulating the local DNA structure to change transcription output, including TATA-binding protein, chromatin remodelers, and, importantly, the nucleosome core particle itself. Methods Plasmid construction DNA fragments encoding B. subtilis BmrR and σA were amplified from B. subtilis genomic DNA, and cloned into pTolo-EX5 (ToloBio Inc.) or pET-28a, respectively, using homologous recombination (Novoprotein). The pEASY/Pbmr was constructed by inserting the promoter region (−50 to +50) of bmr gene (amplified from B. subtilis genomic DNA using primers: forward primer, 5′-TTTGCAAATCCGTTGACTCTCCCC-3′; and reverse primer, 5′-AAATAAAAAGGCCTGCGATTACCAGCAGGCCTTAAGGTAATATTTTTCTTCTCCATATGAC-3′ (with the tR2 terminator sequence underlined)) into the pEASY-blunt vector (Transgen Biotech). The detailed primer sequences are listed in Supplementary Table 3. Bacillus subtilis BmrR The expression of Bs-BmrR was induced by 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 18 °C for 14 h in E. coli BL21(DE3) cells carrying pTolo-EX5-Bs-BmrR. The cells were lysed in lysis buffer A (50 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 5% (v/v) glycerol, 5 mM β-mercaptoethanol, protease inhibitor cocktail (Bimake.com Inc.)) using an Avestin EmulsiFlex-C3 cell disrupter (Avestin Inc.). The Bs-BmrR were enriched by a gravity column packed with 2 ml Ni-NTA agarose (Smart Life Sciences Inc.), washed with lysis buffer A containing 20 mM imidazole, and eluted with lysis buffer A containing 300 mM imidazole. The eluted fractions were treated with TEV protease and dialyzed overnight in a dialysis buffer (50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 5% (v/v) glycerol, 5 mM β-mercaptoethanol). The sample was reloaded onto a Ni-NTA column to remove the impurity. The fraction containing Bs-BmrR were further loaded onto a Heparin column (HiTrap Heparin HP 5 ml column, GE Healthcare Life Sciences), and eluted with a salt gradient of buffer A (50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 5% (v/v) glycerol, 1 mM dithiothreitol (DTT)) and buffer B (50 mM Tris-HCl, pH 8.0, 1 M NaCl, 5% (v/v) glycerol, 1 mM DTT). The elute fractions containing target proteins were collected, concentrated to 2 mg/ml, and stored at −80 °C. Bacillus subtilis σA The E. coli BL21(DE3) cells carrying pET-28a-σA were cultured at 37 °C in LB medium to OD600 of 0.6–0.8. Protein expression was induced by 0.5 mM IPTG at 18 °C for 14 h. Cells were harvested and pellets were suspended in lysis buffer B (50 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 5% (v/v) glycerol, 5 mM β-mercaptoethanol, protease inhibitor cocktail (Bimake.com Inc.)) and lysed using an Avestin EmulsiFlex-C3 cell disrupter (Avestin Inc.). The supernatant of the lysate was loaded onto a gravity column packed with 2 ml Ni-NTA agarose (Smart Lifesciences Inc., China), which was washed with lysis buffer B containing 20 mM imidazole and eluted with lysis buffer B containing 300 mM imidazole. The eluted fractions were dialyzed overnight in the dialysis buffer as described above. The sample were loaded onto a Heparin column (HiTrap Heparin HP 5 ml column, GE Healthcare Life Sciences), and eluted with a salt gradient of buffer A (50 mM Tris-HCl, pH 8.0, 0.05 M NaCl, 5% (v/v) glycerol, 1 mM DTT) and buffer B (50 mM Tris-HCl, pH 8.0, 1 M NaCl, 5% (v/v) glycerol, 1 mM DTT). The elute fractions containing Bs σA were concentrated to ~4 mg/ml and stored at −80 °C. Bacillus subtilis RNAP holoenzyme Bacillus subtilis RNAP holoenzyme was isolated from B. subtilis (strain 168). The cell pellet was resuspended in the lysis buffer C (40 mM Tris-HCl, pH 7.7, 200 mM NaCl, 5% glycerol, 2 mM EDTA, 2 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail (Biomake.com Inc.)) and lysed using a cell disrupter (Avestin EmulsiFlex-C3). The supernatant was precipitated with polyethylenimine (0.7% final) at 4 °C. The pellet was washed and then resuspended with TGED buffer (10 mM Tris-HCl, pH 7.7, 5% glycerol, 1 mM DTT, and 2 mM EDTA) with an additional 1 M NaCl. The supernatant was precipitated by ammonium sulfate (35 g/ml final) and the resulting pellet was collected and dissolved with TGED buffer. The supernatant was loaded onto a Heparin column (HiTrap Heparin HP 5 ml column, GE Healthcare Life Sciences), and eluted with a salt gradient of buffer HPA (40 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 5% (v/v) glycerol, 1 mM DTT, and 0.1 mM EDTA) and buffer HPB (50 mM Tris-HCl, pH 8.0, 1 M NaCl, 5% (v/v) glycerol, 1 mM DTT, and 0.1 mM EDTA). The eluted fractions were further loaded onto a Mono Q column (Mono Q 10/100 GL, GE Healthcare Life Sciences), followed by a salt gradient of buffer QA (40 mM Tris-HCl, pH 7.7, 100 mM NaCl, 5% (v/v) glycerol, 1 mM DTT, 0.1 mM EDTA) and buffer QB (40 mM Tris-HCl, pH 7.7, 600 mM NaCl, 5% (v/v) glycerol, 1 mM DTT, 0.1 mM EDTA). The fractions containing target proteins were collected, concentrated to 5 mg/ml, and stored at −80 °C. The yield of RNAP holoenzyme is 0.4 mg/l. Assembly of B. subtilis BmrR-TAC Nucleic-acid scaffold for B. subtilis BmrR-TAC was prepared from synthetic oligos: non-template-strand DNA (0.5 mM final; Sangon Biotech), template-strand DNA (0.6 mM final; Sangon Biotech), and RNA (0.75 mM final; GenScript Biotech Corp.) in an annealing buffer (5 mM Tris-HCl, pH 8.0, 200 mM NaCl, and 10 mM MgCl2) by an annealing procedure (95 °C, 5 min, followed by 2 °C step cooling to 25 °C). Bs RNAP-σA holoenzyme, Bs σA, Bs-BmrR, and the nucleic-acid scaffold were incubated in a 1:0.5:4:1.3 molar ratio in the presence of 0.5 mM TPP at 4 °C for 1 h (The additional 0.5 molar ratio of Bs σA is included in the mixture to convert possible contamination of RNAP core enzyme to Bs RNAP-σA holoenzyme.) The mixture was applied to a Superose 6 10/300 GL column (GE Healthcare Life Sciences) equilibrated in 10 mM HEPES, pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.5 mM TPP, and 3 mM DTT. Fractions containing B. subtilis BmrR-TAC were collected and concentrated to ~19 mg/ml. Cryo-EM structure determination The freshly purified B. subtilis BmrR-TAC at 15 mg/ml was mixed with 3-([3-cholamidopropyl] dimethylammonio)−2-hydroxy-1-propanesulfonate (CHAPSO, 8 mM final; Hampton Research Inc.) prior to grid preparation. The mixture (3 μl) was loaded on the glow-discharged (120 s) C-flat CF-1.2/1.3 400 mesh holey carbon grids and excess samples were blotted for 8 s with a blot force of −2 at 10 °C and 95% humidity in the chamber of Vitrobot Mark IV (Thermo Fisher Scientific). Subsequently, the grids were vitrified by plunging into liquid ethane. Cryo-EM imaging was performed on a Titan Krios equipped with a Gatan K2 Summit direct electron detector. Data were collected at a nominal magnification of ×22,500 (1.0 Å/pixel) with a dose rate of 8 electrons/pixel/s on sample (~7.4 electrons/pixel/s on detector). A total of 2226 images were recorded using Serial EM with super mode for 7.6 s exposures in 38 subframes to give a total dose of 60.8 electrons/Å2 with defocus range of −1.5 to −2.5 μm. Frames in individual movies were aligned using MotionCor246, and Contrast-transfer-function estimations were performed using CTFFIND447. Auto-picked 494,295 particles were manually inspected and subjected to 2D classification specifying 100 classes in RELION 3.048. Poorly populated classes were removed, resulting in a dataset of 293,724 particles. We use a 40 Å low-pass-filtered map calculated from the cryo-EM structure of E. coli CueR-TAC (PDB: 6LDI) as the starting reference model for 3D classification. Among the 3D classes, the best-resolved class containing 118,891 particles. The dataset of 118,891 particles calculated through 3D auto-refinement, CTF-refinement, Bayesian polishing, and post-processing in RELION 3.0. Subsequent masked 3D classification (N = 2, without alignment) was performed on 118,891 particle projection by subtracting density outside the BmrR, upstream DNA, and σA. The best-resolved class containing 103,226 particles and calculated through 3D auto-refinement, post-processing in RELION 3.0. The Gold-standard Fourier-shell-correlation analysis indicated a mean map resolution of 4.4 Å. The cryo-EM structure of E. coli CueR-TAC (PDB: 6LDI), crystal structure of Bs-BmrR–DNA (PDB:3Q3D), and crystal structure of Geobacillus stearothermophilus YkzG (PDB:4NJC) were manually fit into the cryo-EM density map in Coot49, followed by adjustment of main- and side-chain conformations in Coot, and real space refined using Phenix50. Fluorescence-detected in vitro transcription assay In light of the capability of bulky noncanonical primer-dependent transcription initiation51, we developed a fluorescence-detected in vitro transcription assay using a 5′ Cy3-labeled CGA RNA primer. Bacillus subtilis BmrR-TAC was assembled as above, except that RNA primer was omitted. The reactions mixtures (20 μl) containing 1 μM B. subtilis BmrR-TAC and 1 μM 5′ Cy3-labeled CGA RNA were pre-incubated on ice in transcription buffer (40 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM MgCl2, 12% glycerol, 50 μg/ml bovine serum albumin, 1 mM DTT). The reactions were allowed by the addition of 1 μl 20 μM CTP (final concentration, 1 μM) and further incubation at 37 °C for 30 min and subsequently terminated by the addition of 5 μl stop buffer (8 M urea, 20 mM EDTA, and 0.025% xylene cyanol). The reaction mixtures were heated at 95 °C for 5 min, transferred to ice for 5 min, and electrophoresed on 20% TBE-Urea polyacrylamide gels. The signals were scanned on a fluorescence imager using a 532 nm laser and 570 ± 20 nm filter (Typhoon; GE Healthcare Inc.). Radiochemical in vitro transcription assay Templates for in vitro transcription assay were amplified by PCR using pEASY/P-bmr as a template and M13 primers. Reactions mixtures (20 μl) containing 50 nM B. subtilis RNAP holoenzyme, 200 nM σA, 400 nM BmrR, 200 μM TPP, and 50 nM promoter DNA were pre-incubated at 37 °C for 15 min in transcription buffer. The reactions were allowed by addition of 1 μl NTP mixture to reach a final concentration of 100 μM CTP, 100 μM GTP, 100 μM ATP, 100 μM CTP, and 100 μM [α-32P]UTP (0.04 Bq/fmol) and further incubation at 37 °C for 15 min, and terminated by the addition of 5 μl stop buffer (8 M urea, 20 mM EDTA, 0.025% xylene cyanol, and 0.025% bromophenol blue). The reaction mixtures were heated at 95 °C for 5 min, transferred to ice for 5 min, electrophoresed on 15% TBE-Urea polyacrylamide gels, and analyzed by phosphor imaging (Typhoon; GE Healthcare Inc.). DNA structure analysis The groove widths were measured with w3DNA (http://web.x3dna.org/), and the data were plotted in SigmaPlot14.0 (Systat Software Inc.). The DNA-distortion angles (kinks) were measured with UCSF Chimera. The centroid positions were created at the base-pair center of sites −38, −35, −31, −24, −18, and −13, and the angles between the centroid positions were measured using the Angles/Torsion function. Gel-shift assay Nucleic-acid scaffolds (sequences are listed in Supplementary Fig. 1a) for the gel-shift assay were prepared as above. Reaction mixtures contained (4 μl): 0.75 μM B. subtilis RNAP holoenzyme, 0.5 μM Pbmr-2 or Pbmr-wt1 promoter DNA, 0 or 2 μM BmrR, 0 or 500 μM TPP in 10 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, and 3 mM DTT. Reaction mixtures were incubated for 1 h at room temperature and followed by heparin challenge (100 μg/ml; final concentration) when indicated. The complexes were separated by 5% TBE gel in the TBE buffer (90 mM Tris-borate, pH 8.0, and 2 mM EDTA), stained using SYBR-Gold, and analyzed by Tanon-2500 (Tanon Science & Technology Co., Ltd). Reporting summary Further information on experimental design is available in the Nature Research Reporting Summary linked to this paper. Supplementary information Supplementary Information Peer Review File Reporting Summary Source data Source Data Peer review information Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. These authors contributed equally: Chengli Fang, Linyu Li. Supplementary information Supplementary information is available for this paper at 10.1038/s41467-020-20134-y. Acknowledgements The work was supported by the Strategic Priority Research Program of the CAS to Y.Z. (XDB29020000), the National Key Research and Development Program of China to Y.Z. (2018YFA0900701), the Shanghai Science and technology innovation program to Y.Z. (19JC1415900), the National Natural Science Foundation of China to Y.Z. (31822001 and 31970040) and to Q.L. (81973399), and the Leading Science Key Research Program of CAS to Y.Z. (QYZDB-SSW-SMC005), and the Shanghai “Rising Stars of Medical Talent” Youth Development Program to Q.L. (Youth Medical Talents—Clinical Pharmacist Program), and grants from the National Institutes of Health of the United States to T.V.O. (GM038784-31 and CA193419). We thank Liangliang Kong and Fangfang Wang at the cryo-EM center of the National Center for Protein Science Shanghai (NCPSS) for assistance with data collection. Author contributions L.L. purified endogenous B. subtilis BmrR, assembled BmrR-TAC complex, and performed the gel-shift and transcription assays. C.F. collected the cryo-EM data, calculated the cryo-EM maps, and completed the model building. Y. Zhao assisted in complex assembly, X.W. and L.Y. assisted in cryo-EM data collection and structure determination. M.Z., X.S., Q.L., and Y. Zhang designed and supervised the project. T.V.O., S.J.P., Q.L., and Y. Zhang provided mechanistic interpretation. Q.L. and Y. Zhang wrote and edited the manuscript. Data availability The final coordinate and cryo-EM map were deposited into the protein data bank and the electron microscopy data bank with accession codes of 7CKQ and 30390, respectively. Other data are available from the corresponding authors upon reasonable request. Source data are provided with this paper. Competing interests The authors declare no competing interests. ==== Refs References 1. Browning DF Busby SJ Local and global regulation of transcription initiation in bacteria Nat. Rev. Microbiol. 2016 14 638 650 10.1038/nrmicro.2016.103 27498839 2. Browning DF Butala M Busby SJW Bacterial transcription factors: regulation by pick “N” mix J. Mol. Biol. 2019 431 4067 4077 10.1016/j.jmb.2019.04.011 30998934 3. Busby SJW Transcription activation in bacteria: ancient and modern Microbiology 2019 165 386 395 10.1099/mic.0.000783 30775965 4. Feng Y Zhang Y Ebright RH Structural basis of transcription activation Science 2016 352 1330 1333 10.1126/science.aaf4417 27284196 5. Liu B Hong C Huang RK Yu Z Steitz TA Structural basis of bacterial transcription activation Science 2017 358 947 951 10.1126/science.aao1923 29146813 6. Brown NL Stoyanov JV Kidd SP Hobman JL The MerR family of transcriptional regulators FEMS Microbiol. Rev. 2003 27 145 163 10.1016/S0168-6445(03)00051-2 12829265 7. Heldwein EE Brennan RG Crystal structure of the transcription activator BmrR bound to DNA and a drug Nature 2001 409 378 382 10.1038/35053138 11201751 8. Philips SJ Allosteric transcriptional regulation via changes in the overall topology of the core promoter Science 2015 349 877 881 10.1126/science.aaa9809 26293965 9. Ansari AZ Chael ML O’Halloran TV Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR Nature 1992 355 87 89 10.1038/355087a0 1731201 10. Ansari AZ Bradner JE O’Halloran TV DNA-bend modulation in a repressor-to-activator switching mechanism Nature 1995 374 371 375 10.1038/374370a0 7885478 11. Martell DJ Metalloregulator CueR biases RNA polymerase’s kinetic sampling of dead-end or open complex to repress or activate transcription Proc. Natl Acad. Sci. USA 2015 112 13467 13472 10.1073/pnas.1515231112 26483469 12. Liu X Selective cadmium regulation mediated by a cooperative binding mechanism in CadR Proc. Natl Acad. Sci. USA 2019 116 20398 20403 10.1073/pnas.1908610116 31548408 13. Changela A Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR Science 2003 301 1383 1387 10.1126/science.1085950 12958362 14. Brocklehurst KR ZntR is a Zn(II)-responsive MerR-like transcriptional regulator of zntA in Escherichia coli Mol. Microbiol. 1999 31 893 902 10.1046/j.1365-2958.1999.01229.x 10048032 15. Borremans B Hobman JL Provoost A Brown NL van Der Lelie D Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34 J. Bacteriol. 2001 183 5651 5658 10.1128/JB.183.19.5651-5658.2001 11544228 16. Lee SW Glickmann E Cooksey DA Chromosomal locus for cadmium resistance in Pseudomonas putida consisting of a cadmium-transporting ATPase and a MerR family response regulator Appl. Environ. Microbiol. 2001 67 1437 1444 10.1128/AEM.67.4.1437-1444.2001 11282588 17. Rutherford JC Cavet JS Robinson NJ Cobalt-dependent transcriptional switching by a dual-effector MerR-like protein regulates a cobalt-exporting variant CPx-type ATPase J. Biol. Chem. 1999 274 25827 25832 10.1074/jbc.274.36.25827 10464323 18. Watanabe S Kita A Kobayashi K Miki K Crystal structure of the [2Fe-2S] oxidative-stress sensor SoxR bound to DNA Proc. Natl Acad. Sci. USA 2008 105 4121 4126 10.1073/pnas.0709188105 18334645 19. Ahmed M Borsch CM Taylor SS Vazquez-Laslop N Neyfakh AA A protein that activates expression of a multidrug efflux transporter upon binding the transporter substrates J. Biol. Chem. 1994 269 28506 28513 7961792 20. Ahmed M Two highly similar multidrug transporters of Bacillus subtilis whose expression is differentially regulated J. Bacteriol. 1995 177 3904 3910 10.1128/JB.177.14.3904-3910.1995 7608059 21. Baranova NN Danchin A Neyfakh AA Mta, a global MerR-type regulator of the Bacillus subtilis multidrug-efflux transporters Mol. Microbiol. 1999 31 1549 1559 10.1046/j.1365-2958.1999.01301.x 10200972 22. O’Halloran TV Frantz B Shin MK Ralston DM Wright JG The MerR heavy metal receptor mediates positive activation in a topologically novel transcription complex Cell 1989 56 119 129 10.1016/0092-8674(89)90990-2 2910495 23. Zheleznova EE Markham PN Neyfakh AA Brennan RG Structural basis of multidrug recognition by BmrR, a transcription activator of a multidrug transporter Cell 1999 96 353 362 10.1016/S0092-8674(00)80548-6 10025401 24. Bachas S Eginton C Gunio D Wade H Structural contributions to multidrug recognition in the multidrug resistance (MDR) gene regulator, BmrR Proc. Natl Acad. Sci. USA 2011 108 11046 11051 10.1073/pnas.1104850108 21690368 25. Kumaraswami M Newberry KJ Brennan RG Conformational plasticity of the coiled-coil domain of BmrR is required for bmr operator binding: the structure of unliganded BmrR J. Mol. Biol. 2010 398 264 275 10.1016/j.jmb.2010.03.011 20230832 26. Markham PN Ahmed M Neyfakh AA The drug-binding activity of the multidrug-responding transcriptional regulator BmrR resides in its C-terminal domain J. Bacteriol. 1996 178 1473 1475 10.1128/JB.178.5.1473-1475.1996 8631728 27. Markham PN LoGuidice J Neyfakh AA Broad ligand specificity of the transcriptional regulator of the Bacillus subtilis multidrug transporter Bmr Biochem. Biophys. Res. Commun. 1997 239 269 272 10.1006/bbrc.1997.7467 9345308 28. Bachas S Kohrs B Wade H Charge is major determinant of activation of the ligand-responsive multidrug resistance gene regulator, BmrR ChemMedChem 2016 11 1038 1041 10.1002/cmdc.201600059 27010425 29. Newberry KJ Structures of BmrR-drug complexes reveal a rigid multidrug binding pocket and transcription activation through tyrosine expulsion J. Biol. Chem. 2008 283 26795 26804 10.1074/jbc.M804191200 18658145 30. Newberry KJ Brennan RG The structural mechanism for transcription activation by MerR family member multidrug transporter activation, N terminus J. Biol. Chem. 2004 279 20356 20362 10.1074/jbc.M400960200 14985361 31. Narayanan A Cryo-EM structure of Escherichia coli sigma(70) RNA polymerase and promoter DNA complex revealed a role of sigma non-conserved region during the open complex formation J. Biol. Chem. 2018 293 7367 7375 10.1074/jbc.RA118.002161 29581236 32. Bae, B., Feklistov, A., Lass-Napiorkowska, A., Landick, R. & Darst, S. A. Structure of a bacterial RNA polymerase holoenzyme open promoter complex. eLife4, e08504 (2015). 33. Boyaci H Chen J Jansen R Darst SA Campbell EA Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding Nature 2019 565 382 385 10.1038/s41586-018-0840-5 30626968 34. Hubin, E. A. et al. Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA. eLife6, e22520 (2017). 35. Spiegelman GB Hiatt WR Whiteley HR Role of the 21,000 molecular weight polypeptide of Bacillus subtilis RNA polymerase in RNA synthesis J. Biol. Chem. 1978 253 1756 1765 416017 36. Keller AN epsilon, a new subunit of RNA polymerase found in Gram-positive bacteria J. Bacteriol. 2014 196 3622 3632 10.1128/JB.02020-14 25092033 37. Bae B Phage T7 Gp2 inhibition of Escherichia coli RNA polymerase involves misappropriation of sigma70 domain 1.1 Proc. Natl Acad. Sci. USA 2013 110 19772 19777 10.1073/pnas.1314576110 24218560 38. Campbell EA Structure of the bacterial RNA polymerase promoter specificity sigma subunit Mol. Cell 2002 9 527 539 10.1016/S1097-2765(02)00470-7 11931761 39. Zhang Y Structural basis of transcription initiation Science 2012 338 1076 1080 10.1126/science.1227786 23086998 40. Nikolov DB Crystal structure of a TFIIB-TBP-TATA-element ternary complex Nature 1995 377 119 128 10.1038/377119a0 7675079 41. Shultzaberger RK Chen Z Lewis KA Schneider TD Anatomy of Escherichia coli sigma70 promoters Nucleic Acids Res. 2007 35 771 788 10.1093/nar/gkl956 17189297 42. Chen J Stepwise promoter melting by bacterial RNA polymerase Mol. Cell 2020 78 275 288 e276 10.1016/j.molcel.2020.02.017 32160514 43. Fang, C. et al. CueR activates transcription through a DNA distortion mechanism. Nat. Chem. Biol.10.1038/s41589-020-00653-x (2020). 44. Zuo Y Steitz TA Crystal structures of the E. coli transcription initiation complexes with a complete bubble Mol. Cell 2015 58 534 540 10.1016/j.molcel.2015.03.010 25866247 45. Kulkarni RD Summers AO MerR cross-links to the alpha, beta, and sigma 70 subunits of RNA polymerase in the preinitiation complex at the merTPCAD promoter Biochemistry 1999 38 3362 3368 10.1021/bi982814m 10079080 46. Zheng SQ MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy Nat. Methods 2017 14 331 332 28250466 47. Rohou A Grigorieff N CTFFIND4: fast and accurate defocus estimation from electron micrographs J. Struct. Biol. 2015 192 216 221 10.1016/j.jsb.2015.08.008 26278980 48. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife7, e42166 (2018). 49. Emsley P Cowtan K Coot: model-building tools for molecular graphics Acta Crystallogr. D 2004 60 2126 2132 10.1107/S0907444904019158 15572765 50. Adams PD PHENIX: a comprehensive Python-based system for macromolecular structure solution Acta Crystallogr. D 2010 66 213 221 10.1107/S0907444909052925 20124702 51. Bird JG The mechanism of RNA 5’ capping with NAD+, NADH and desphospho-CoA Nature 2016 535 444 447 10.1038/nature18622 27383794