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Richard H. Ebright - One of the best experts on this subject based on the ideXlab platform.
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Transcription Initiation at a consensus bacterial promoter proceeds via a bind unwind load and lock mechanism
bioRxiv, 2021Co-Authors: Abhishek Mazumder, Richard H. Ebright, Achillefs N. KapanidisAbstract:Abstract Transcription Initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, “bind-unwind-load-and-lock,” model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters. Significance statement Transcription Initiation, the first step and most important step in gene expression for all organisms, involves unwinding of promoter DNA by RNA polymerase (RNAP) to form an open complex (RPo); this step also underpins Transcriptional regulation and serves as an antibiotic target. Despite decades of research, the mechanism of promoter DNA unwinding has remained unresolved. Here, we solve this puzzle by using single-molecule fluorescence to directly monitor conformational changes in the promoter DNA and RNAP in real time during RPo formation. We show that RPo forms via a “bind-unwind-load-and-lock” mechanism, where the promoter unwinds outside the RNAP cleft, the unwound template DNA loads into the cleft, and RNAP “locks” the template DNA in place by closing the RNAP clamp module.
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structural basis of ecf sigma factor dependent Transcription Initiation
Nature Communications, 2019Co-Authors: David Degen, Sukhendu Mandal, Yu Feng, Kalyan Das, Wei Lin, Min Sung Cho, Richard H. EbrightAbstract:Extracytoplasmic (ECF) σ factors, the largest class of alternative σ factors, are related to primary σ factors, but have simpler structures, comprising only two of six conserved functional modules in primary σ factors: region 2 (σR2) and region 4 (σR4). Here, we report crystal structures of Transcription Initiation complexes containing Mycobacterium tuberculosis RNA polymerase (RNAP), M. tuberculosis ECF σ factor σL, and promoter DNA. The structures show that σR2 and σR4 of the ECF σ factor occupy the same sites on RNAP as in primary σ factors, show that the connector between σR2 and σR4 of the ECF σ factor–although shorter and unrelated in sequence–follows the same path through RNAP as in primary σ factors, and show that the ECF σ factor uses the same strategy to bind and unwind promoter DNA as primary σ factors. The results define protein-protein and protein-DNA interactions involved in ECF-σ-factor-dependent Transcription Initiation. No structural data have been available for RNA polymerase holoenzymes or Transcription Initiation complexes that contain extracytoplasmic σ factors. Here the authors report the crystal structures of Transcription Initiation complexes comprising Mycobacterium tuberculosis RNA polymerase, extracytoplasmic σ factor σL and promoter DNA.
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Structural basis of ECF-sigma-factor-dependent Transcription Initiation.
bioRxiv, 2018Co-Authors: Sukhendu Mandal, David Degen, Yu Feng, Richard H. EbrightAbstract:Extracytoplasmic (ECF) sigma factors, the largest class of alternative sigma factors, are related to primary sigma factors, but have simpler structures, comprising only two of the six conserved functional modules present in primary sigma factors: region 2 (sigmaR2) and region 4 (sigmaR4). Here, we report crystal structures of Transcription Initiation complexes containing Mycobacterium tuberculosis RNA polymerase (RNAP), M. tuberculosis ECF sigma factor sigma-L, and promoter DNA. The structures show that sigmaR2 and sigmaR4 of the ECF sigma factor occupy the same sites on RNAP as in primary sigma factors, show that the connector between sigmaR2 and sigmaR4 of the ECF sigma factor--although unrelated in sequence--follows the same path through RNAP as in primary sigma factors, and show that the ECF sigma factor uses the same strategy to bind and unwind promoter DNA as primary sigma factors. The results define protein-protein and protein-DNA interactions involved in ECF-sigma-factor-dependent Transcription Initiation.
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rna capping by Transcription Initiation with non canonical initiating nucleotides ncins determination of relative efficiencies of Transcription Initiation with ncins and ntps
Bio-protocol, 2017Co-Authors: Jeremy G Bird, Bryce E Nickels, Richard H. EbrightAbstract:It recently has been established that adenine-containing cofactors, including nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), and 3'-desphospho-coenzyme A (dpCoA), can serve as 'non-canonical initiating nucleotides' (NCINs) for Transcription Initiation by bacterial and eukaryotic cellular RNA polymerases (RNAPs) and that the efficiency of the reaction is determined by promoter sequence (Bird et al., 2016). Here we describe a protocol to quantify the relative efficiencies of Transcription Initiation using an NCIN vs. Transcription Initiation using a nucleoside triphosphate (NTP) for a given promoter sequence.
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the Initiation factor tfe and the elongation factor spt4 5 compete for the rnap clamp during Transcription Initiation and elongation
Molecular Cell, 2011Co-Authors: Dina Grohmann, Richard H. Ebright, Julia Nagy, Anirban Chakraborty, Daniel Klose, Daniel Fielden, Jens Michaelis, Finn WernerAbstract:TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during Transcription Initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal Transcription Initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the Transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed Transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP “clamp” region as a regulatory hot spot for both Transcription Initiation and Transcription elongation.
Yu Zhang - One of the best experts on this subject based on the ideXlab platform.
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structures and mechanism of Transcription Initiation by bacterial ecf factors
Nucleic Acids Research, 2019Co-Authors: Chengli Fang, Yu Feng, Liqiang Shen, Jing Shi, Sheng Wang, Yu ZhangAbstract:Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 A of Escherichia coli Transcription Initiation complex comprising σE-the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 A of Mycobacterium tuberculosis Transcription Initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated Transcription Initiation.
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structural basis for Transcription Initiation by bacterial ecf sigma factors
Nature Communications, 2019Co-Authors: Lingting Li, Chengli Fang, Ningning Zhuang, Tiantian Wang, Yu ZhangAbstract:Bacterial RNA polymerase employs extra-cytoplasmic function (ECF) σ factors to regulate context-specific gene expression programs. Despite being the most abundant and divergent σ factor class, the structural basis of ECF σ factor-mediated Transcription Initiation remains unknown. Here, we determine a crystal structure of Mycobacterium tuberculosis (Mtb) RNAP holoenzyme comprising an RNAP core enzyme and the ECF σ factor σH (σH-RNAP) at 2.7 A, and solve another crystal structure of a Transcription Initiation complex of Mtb σH-RNAP (σH-RPo) comprising promoter DNA and an RNA primer at 2.8 A. The two structures together reveal the interactions between σH and RNAP that are essential for σH-RNAP holoenzyme assembly as well as the interactions between σH-RNAP and promoter DNA responsible for stringent promoter recognition and for promoter unwinding. Our study establishes that ECF σ factors and primary σ factors employ distinct mechanisms for promoter recognition and for promoter unwinding. Extra-cytoplasmic function (ECF) σ factors allow bacteria to rapidly respond to stress conditions. Here the authors provide insights into the mechanism of bacterial stress-induced Transcription by determining the crystal structures of the M. tuberculosis ECF σ factor bound RNA Polymerase (RNAP) holoenzyme and the ECF σ factor-RNAP Transcription Initiation complex.
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Structural basis for Transcription Initiation by bacterial ECF σ factors
Nature Publishing Group, 2019Co-Authors: Chengli Fang, Ningning Zhuang, Tiantian Wang, Yu ZhangAbstract:Extra-cytoplasmic function (ECF) σ factors allow bacteria to rapidly respond to stress conditions. Here the authors provide insights into the mechanism of bacterial stress-induced Transcription by determining the crystal structures of the M. tuberculosis ECF σ factor bound RNA Polymerase (RNAP) holoenzyme and the ECF σ factor-RNAP Transcription Initiation complex
Wei Lin - One of the best experts on this subject based on the ideXlab platform.
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structural basis of ecf sigma factor dependent Transcription Initiation
Nature Communications, 2019Co-Authors: David Degen, Sukhendu Mandal, Yu Feng, Kalyan Das, Wei Lin, Min Sung Cho, Richard H. EbrightAbstract:Extracytoplasmic (ECF) σ factors, the largest class of alternative σ factors, are related to primary σ factors, but have simpler structures, comprising only two of six conserved functional modules in primary σ factors: region 2 (σR2) and region 4 (σR4). Here, we report crystal structures of Transcription Initiation complexes containing Mycobacterium tuberculosis RNA polymerase (RNAP), M. tuberculosis ECF σ factor σL, and promoter DNA. The structures show that σR2 and σR4 of the ECF σ factor occupy the same sites on RNAP as in primary σ factors, show that the connector between σR2 and σR4 of the ECF σ factor–although shorter and unrelated in sequence–follows the same path through RNAP as in primary σ factors, and show that the ECF σ factor uses the same strategy to bind and unwind promoter DNA as primary σ factors. The results define protein-protein and protein-DNA interactions involved in ECF-σ-factor-dependent Transcription Initiation. No structural data have been available for RNA polymerase holoenzymes or Transcription Initiation complexes that contain extracytoplasmic σ factors. Here the authors report the crystal structures of Transcription Initiation complexes comprising Mycobacterium tuberculosis RNA polymerase, extracytoplasmic σ factor σL and promoter DNA.
Yu Feng - One of the best experts on this subject based on the ideXlab platform.
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structures and mechanism of Transcription Initiation by bacterial ecf factors
Nucleic Acids Research, 2019Co-Authors: Chengli Fang, Yu Feng, Liqiang Shen, Jing Shi, Sheng Wang, Yu ZhangAbstract:Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 A of Escherichia coli Transcription Initiation complex comprising σE-the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 A of Mycobacterium tuberculosis Transcription Initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated Transcription Initiation.
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structural basis of ecf sigma factor dependent Transcription Initiation
Nature Communications, 2019Co-Authors: David Degen, Sukhendu Mandal, Yu Feng, Kalyan Das, Wei Lin, Min Sung Cho, Richard H. EbrightAbstract:Extracytoplasmic (ECF) σ factors, the largest class of alternative σ factors, are related to primary σ factors, but have simpler structures, comprising only two of six conserved functional modules in primary σ factors: region 2 (σR2) and region 4 (σR4). Here, we report crystal structures of Transcription Initiation complexes containing Mycobacterium tuberculosis RNA polymerase (RNAP), M. tuberculosis ECF σ factor σL, and promoter DNA. The structures show that σR2 and σR4 of the ECF σ factor occupy the same sites on RNAP as in primary σ factors, show that the connector between σR2 and σR4 of the ECF σ factor–although shorter and unrelated in sequence–follows the same path through RNAP as in primary σ factors, and show that the ECF σ factor uses the same strategy to bind and unwind promoter DNA as primary σ factors. The results define protein-protein and protein-DNA interactions involved in ECF-σ-factor-dependent Transcription Initiation. No structural data have been available for RNA polymerase holoenzymes or Transcription Initiation complexes that contain extracytoplasmic σ factors. Here the authors report the crystal structures of Transcription Initiation complexes comprising Mycobacterium tuberculosis RNA polymerase, extracytoplasmic σ factor σL and promoter DNA.
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Structural basis of ECF-sigma-factor-dependent Transcription Initiation
'Springer Science and Business Media LLC', 2019Co-Authors: Lin Wei, Yu Feng, Das Kalyan, Mandal Sukhendu, Degen David, Cho, Min Sung, Ebright, Richard HAbstract:Extracytoplasmic (ECF) σ factors, the largest class of alternative σ factors, are related to primary σ factors, but have simpler structures, comprising only two of six conserved functional modules in primary σ factors: region 2 (σR2) and region 4 (σR4). Here, we report crystal structures of Transcription Initiation complexes containing Mycobacterium tuberculosis RNA polymerase (RNAP), M. tuberculosis ECF σ factor σL, and promoter DNA. The structures show that σR2 and σR4 of the ECF σ factor occupy the same sites on RNAP as in primary σ factors, show that the connector between σR2 and σR4 of the ECF σ factor-although shorter and unrelated in sequence-follows the same path through RNAP as in primary σ factors, and show that the ECF σ factor uses the same strategy to bind and unwind promoter DNA as primary σ factors. The results define protein-protein and protein-DNA interactions involved in ECF-σ-factor-dependent Transcription Initiation.status: publishe
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Structural basis of ECF-sigma-factor-dependent Transcription Initiation.
bioRxiv, 2018Co-Authors: Sukhendu Mandal, David Degen, Yu Feng, Richard H. EbrightAbstract:Extracytoplasmic (ECF) sigma factors, the largest class of alternative sigma factors, are related to primary sigma factors, but have simpler structures, comprising only two of the six conserved functional modules present in primary sigma factors: region 2 (sigmaR2) and region 4 (sigmaR4). Here, we report crystal structures of Transcription Initiation complexes containing Mycobacterium tuberculosis RNA polymerase (RNAP), M. tuberculosis ECF sigma factor sigma-L, and promoter DNA. The structures show that sigmaR2 and sigmaR4 of the ECF sigma factor occupy the same sites on RNAP as in primary sigma factors, show that the connector between sigmaR2 and sigmaR4 of the ECF sigma factor--although unrelated in sequence--follows the same path through RNAP as in primary sigma factors, and show that the ECF sigma factor uses the same strategy to bind and unwind promoter DNA as primary sigma factors. The results define protein-protein and protein-DNA interactions involved in ECF-sigma-factor-dependent Transcription Initiation.
E M Flynn - One of the best experts on this subject based on the ideXlab platform.
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gne 371 a potent and selective chemical probe for the second bromodomains of human Transcription Initiation factor tfiid subunit 1 and Transcription Initiation factor tfiid subunit 1 like
Journal of Medicinal Chemistry, 2018Co-Authors: Shumei Wang, Vickie Tsui, Terry Crawford, Daniel J Burdick, Maureen Beresini, Martin Duplessis, Alexandre Cote, James E Audia, Richard D. Cummings, E M FlynnAbstract:The biological functions of the dual bromodomains of human Transcription-Initiation-factor TFIID subunit 1 (TAF1(1,2)) remain unknown, although TAF1 has been identified as a potential target for oncology research. Here, we describe the discovery of a potent and selective in vitro tool compound for TAF1(2), starting from a previously reported lead. A cocrystal structure of lead compound 2 bound to TAF1(2) enabled structure-based design and structure–activity-relationship studies that ultimately led to our in vitro tool compound, 27 (GNE-371). Compound 27 binds TAF1(2) with an IC50 of 10 nM while maintaining excellent selectivity over other bromodomain-family members. Compound 27 is also active in a cellular-TAF1(2) target-engagement assay (IC50 = 38 nM) and exhibits antiproliferative synergy with the BET inhibitor JQ1, suggesting engagement of endogenous TAF1 by 27 and further supporting the use of 27 in mechanistic and target-validation studies.
