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Patrick Cramer - One of the best experts on this subject based on the ideXlab platform.
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Structures of transcription pre-Initiation Complex with TFIIH and Mediator
Nature, 2017Co-Authors: S. Schilbach, M. Hantsche, D. Tegunov, Christian Dienemann, C. Wigge, Henning Urlaub, Patrick CramerAbstract:For the Initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-Initiation Complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC–core Mediator Complex at nominal resolutions of 4.7 A and 5.8 A, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the ‘E-bridge’ helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC–core Mediator Complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II. Cryo-electron microscopy structures of the yeast pre-Initiation Complex (PIC) and its Complex with core Mediator provide insights into the opening of promoter DNA and the Initiation of transcription. To initiate gene transcription, RNA polymerase (Pol) II assembles with general transcription factors on promoter DNA to form the pre-Initiation Complex (PIC). Here, Patrick Cramer and colleagues describe cryo-electron microscopy structures of the yeast PIC and the PIC bound to the core Mediator (cMed) Complex. The latter structure with the general coactivator Mediator has 46 factors, including all those that are essential for transcription Initiation in yeast. The structures reveal the architecture of transcription factor IIH (TFIIH) and suggest how its 'core' and 'kinase' modules might function in promoter opening and Pol II phosphorylation, respectively.
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core mediator structure at 3 4 a extends model of transcription Initiation Complex
Nature, 2017Co-Authors: Kayo Nozawa, Thomas R Schneider, Patrick CramerAbstract:The 3.4 A crystal structure of the 15-subunit core Mediator Complex in yeast. The multiprotein Mediator Complex has an essential role in regulating RNA polymerase II (Pol II) transcription in eukaryotes. Here, Patrick Cramer and colleagues report a 3.4 A crystal structure of the 15-subunit core Mediator Complex in yeast. They combine this with a previously determined cryo-EM structure of the Pol II pre-Initiation Complex to obtain an atomic model of Mediator bound to the pre-Initiation Complex. This model allows insights into the interactions of the head and middle modules of Mediator and provides a framework for understanding how Mediator stimulates Pol II C-terminal domain phosphorylation by TFIIH, a process which triggers productive transcription. Mediator is a multiprotein co-activator that binds the transcription pre-Initiation Complex (PIC) and regulates RNA polymerase (Pol) II1,2,3. The Mediator head and middle modules form the essential core Mediator (cMed)4,5,6, whereas the tail and kinase modules play regulatory roles7. The architecture of Mediator5,8,9,10 and its position on the PIC5 are known, but atomic details are limited to Mediator subComplexes11,12. Here we report the crystal structure of the 15-subunit cMed from Schizosaccharomyces pombe at 3.4 A resolution. The structure shows an unaltered head module13,14,15, and reveals the intricate middle module, which we show is globally required for transcription. Sites of known Mediator mutations cluster at the interface between the head and middle modules, and in terminal regions of the head subunits Med6 (ref. 16) and Med17 (ref. 17) that tether the middle module. The structure led to a model for Saccharomyces cerevisiae cMed that could be combined5 with the 3.6 A cryo-electron microscopy structure of the core PIC (cPIC)18. The resulting atomic model of the cPIC–cMed Complex informs on interactions of the submodules forming the middle module, called beam, knob, plank, connector, and hook. The hook is flexibly linked to Mediator by a conserved hinge19 and contacts the transcription Initiation factor IIH (TFIIH) kinase that phosphorylates the carboxy (C)-terminal domain (CTD) of Pol II and was recently positioned on the PIC20. The hook also contains residues that crosslink to the CTD and reside in a previously described cradle5. These results provide a framework for understanding Mediator function, including its role in stimulating CTD phosphorylation by TFIIH.
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Conserved RNA polymerase II Initiation Complex structure.
Current opinion in structural biology, 2017Co-Authors: M. Hantsche, Patrick CramerAbstract:Recent cryo-electron microscopic studies have arrived at atomic models of the core transcription Initiation Complex comprising RNA polymerase (Pol) II and the basal transcription factors TBP, TFIIA, TFIIB, TFIIE, and TFIIF. A detailed comparison of two independently derived yeast and human core Initiation Complex structures reveals that they are virtually identical, demonstrating the conservation of the basic transcription machinery amongst eukaryotes. The additional factors TFIID, TFIIH, and Mediator have been located on the periphery of the core Initiation Complex, providing the topology of the entire Initiation assembly, which comprises approximately 70 polypeptides with a molecular weight of ∼4 Megadalton.
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Transcription Initiation Complex structures elucidate DNA opening.
Nature, 2016Co-Authors: C. Plaschka, M. Hantsche, Christian Dienemann, C. Burzinski, Jürgen M. Plitzko, Patrick CramerAbstract:Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II Initiation Complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast Initiation Complexes containing closed and open DNA at resolutions of 8.8 A and 3.6 A, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription Initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts.
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Conserved architecture of the core RNA polymerase II Initiation Complex.
Nature communications, 2014Co-Authors: Wolfgang Mühlbacher, M. Hantsche, Sarah Sainsbury, Matthias Hemann, Simon Neyer, Franz Herzog, Patrick CramerAbstract:During transcription Initiation at promoters of protein-coding genes, RNA polymerase (Pol) II assembles with TBP, TFIIB and TFIIF into a conserved core Initiation Complex that recruits additional factors. The core Complex stabilizes open DNA and initiates RNA synthesis, and it is conserved in the Pol I and Pol III transcription systems. Here, we derive the domain architecture of the yeast core pol II Initiation Complex during transcription Initiation. The yeast Complex resembles the human Initiation Complex and reveals that the TFIIF Tfg2 winged helix domain swings over promoter DNA. An 'arm' and a 'charged helix' in TFIIF function in transcription start site selection and initial RNA synthesis, respectively, and apparently extend into the active centre cleft. Our model provides the basis for further structure-function analysis of the entire transcription Initiation Complex.
Gerald R Smith - One of the best experts on this subject based on the ideXlab platform.
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regular articlethe recbcd enzyme Initiation Complex for dna unwinding enzyme positioning and dna opening 1
Journal of Molecular Biology, 1997Co-Authors: Joseph A Farah, Gerald R SmithAbstract:The Escherichia coli RecBCD enzyme unwinds DNA from a free double-stranded DNA end to produce single-stranded DNA intermediates of homologous recombination. In the absence of ATP RecBCD binds to a free DNA end to form an Initiation Complex for DNA unwinding. We studied the structure of these Complexes formed with blunt-ended, 5′-extended, and 3′-extended DNA. Reactivity to the single-stranded DNA-specific reagents KMnO4 and dimethyl sulfate indicated that RecBCD opened, in a Mg2+-dependent manner, the terminal five or six base-pairs in each substrate. Thymine residues located four to six nucleotides from the 5′ end were only partially reactive to KMnO4, suggesting that part of the 5′-terminated strand was partially shielded by the enzyme. DNase I footprinting indicated that the enzyme positions itself relative to the end of the longer of the two strands, although an exception was noted. These results imply flexibility in the ability of RecBCD to open the DNA and position itself for unwinding on DNA with different types of ends. They also imply conformational differences of RecBCD enzyme bound to different types of ends; these conformational differences may be related to those occurring during the unwinding cycle.
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the recbcd enzyme Initiation Complex for dna unwinding enzyme positioning and dna opening
Journal of Molecular Biology, 1997Co-Authors: Joseph A Farah, Gerald R SmithAbstract:Abstract The Escherichia coli RecBCD enzyme unwinds DNA from a free double-stranded DNA end to produce single-stranded DNA intermediates of homologous recombination. In the absence of ATP RecBCD binds to a free DNA end to form an Initiation Complex for DNA unwinding. We studied the structure of these Complexes formed with blunt-ended, 5′-extended, and 3′-extended DNA. Reactivity to the single-stranded DNA-specific reagents KMnO 4 and dimethyl sulfate indicated that RecBCD opened, in a Mg 2+ -dependent manner, the terminal five or six base-pairs in each substrate. Thymine residues located four to six nucleotides from the 5′ end were only partially reactive to KMnO 4 , suggesting that part of the 5′-terminated strand was partially shielded by the enzyme. DNase I footprinting indicated that the enzyme positions itself relative to the end of the longer of the two strands, although an exception was noted. These results imply flexibility in the ability of RecBCD to open the DNA and position itself for unwinding on DNA with different types of ends. They also imply conformational differences of RecBCD enzyme bound to different types of ends; these conformational differences may be related to those occurring during the unwinding cycle.
Eva Nogales - One of the best experts on this subject based on the ideXlab platform.
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Cryo-EM in the study of challenging systems: the human transcription pre-Initiation Complex.
Current opinion in structural biology, 2016Co-Authors: Eva Nogales, Robert K. LouderAbstract:Single particle cryo-Electron Microscopy (cryo-EM) is a technique that allows the structural characterization of macromolecules without the need for crystallization. For certain type of samples that are ideally suited for cryo-EM studies it has been possible to reach high-resolution structures following relatively standard procedures. Other biological systems remain highly challenging, even for cryo-EM. Challenges may involve the scarcity of the sample, poor stability of the Complexes, and most often, the intrinsic flexibility of biological molecules. Among these challenging samples are large eukaryotic transcription Complexes, which suffer from all such shortcomings. Here we report how we have recently tried to overcome those challenges in order to improve our structural understanding of the human transcription pre-Initiation Complex assembly and the transcription Initiation process. Parallel efforts have also been carried out for budding yeast transcription Initiation Complexes, allowing comparisons that establish both the overall conservation and the specific structural differences between the two systems.
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Structure of promoter-bound TFIID and model of human pre-Initiation Complex assembly
Nature, 2016Co-Authors: Robert K. Louder, José Ramón López-blanco, Pablo Chacón, Jie Fang, Yuan He, Eva NogalesAbstract:This paper describes the interaction of the human general transcription factor IID (TFIID) with core promoter DNA. TFIID has a central role in the Initiation of RNA polymerase II dependent transcription by nucleating assembly of the pre-Initiation Complex (PIC) at the promoter. Eva Nogales and colleagues determine a sub-nanometre resolution cryo-electron microscopy structure of TFIID bound to TFIIA and core promoter DNA. They also present a cryo-electron microscopy reconstruction of a fully assembled human PIC lacking TBP-associated factors (TAFs). By superimposing common elements between the TFIID–TFIIA–promoter structure and the TAF-less PIC structure, they propose a structural model for the complete TFIID-based PIC, and provide insights into the role of TFIID in promoter recognition, PIC assembly, and transcription Initiation. The general transcription factor IID (TFIID) plays a central role in the Initiation of RNA polymerase II (Pol II)-dependent transcription by nucleating pre-Initiation Complex (PIC) assembly at the core promoter. TFIID comprises the TATA-binding protein (TBP) and 13 TBP-associated factors (TAF1–13), which specifically interact with a variety of core promoter DNA sequences. Here we present the structure of human TFIID in Complex with TFIIA and core promoter DNA, determined by single-particle cryo-electron microscopy at sub-nanometre resolution. All core promoter elements are contacted by subunits of TFIID, with TAF1 and TAF2 mediating major interactions with the downstream promoter. TFIIA bridges the TBP–TATA Complex with lobe B of TFIID. We also present the cryo-electron microscopy reconstruction of a fully assembled human TAF-less PIC. Superposition of common elements between the two structures provides novel insights into the general role of TFIID in promoter recognition, PIC assembly, and transcription Initiation. A sub-nanometre resolution cryo-EM structure of human TFIID bound to TFIIA and core promoter DNA and a model of the TFIID-based pre-Initiation Complex.
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structure of promoter bound tfiid and model of human pre Initiation Complex assembly
Nature, 2016Co-Authors: Robert K. Louder, Pablo Chacón, Jose Ramon Lopezblanco, Jie Fang, Yuan He, Eva NogalesAbstract:The general transcription factor IID (TFIID) plays a central role in the Initiation of RNA polymerase II (Pol II)-dependent transcription by nucleating pre-Initiation Complex (PIC) assembly at the core promoter. TFIID comprises the TATA-binding protein (TBP) and 13 TBP-associated factors (TAF1-13), which specifically interact with a variety of core promoter DNA sequences. Here we present the structure of human TFIID in Complex with TFIIA and core promoter DNA, determined by single-particle cryo-electron microscopy at sub-nanometre resolution. All core promoter elements are contacted by subunits of TFIID, with TAF1 and TAF2 mediating major interactions with the downstream promoter. TFIIA bridges the TBP-TATA Complex with lobe B of TFIID. We also present the cryo-electron microscopy reconstruction of a fully assembled human TAF-less PIC. Superposition of common elements between the two structures provides novel insights into the general role of TFIID in promoter recognition, PIC assembly, and transcription Initiation.
Joseph A Farah - One of the best experts on this subject based on the ideXlab platform.
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regular articlethe recbcd enzyme Initiation Complex for dna unwinding enzyme positioning and dna opening 1
Journal of Molecular Biology, 1997Co-Authors: Joseph A Farah, Gerald R SmithAbstract:The Escherichia coli RecBCD enzyme unwinds DNA from a free double-stranded DNA end to produce single-stranded DNA intermediates of homologous recombination. In the absence of ATP RecBCD binds to a free DNA end to form an Initiation Complex for DNA unwinding. We studied the structure of these Complexes formed with blunt-ended, 5′-extended, and 3′-extended DNA. Reactivity to the single-stranded DNA-specific reagents KMnO4 and dimethyl sulfate indicated that RecBCD opened, in a Mg2+-dependent manner, the terminal five or six base-pairs in each substrate. Thymine residues located four to six nucleotides from the 5′ end were only partially reactive to KMnO4, suggesting that part of the 5′-terminated strand was partially shielded by the enzyme. DNase I footprinting indicated that the enzyme positions itself relative to the end of the longer of the two strands, although an exception was noted. These results imply flexibility in the ability of RecBCD to open the DNA and position itself for unwinding on DNA with different types of ends. They also imply conformational differences of RecBCD enzyme bound to different types of ends; these conformational differences may be related to those occurring during the unwinding cycle.
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the recbcd enzyme Initiation Complex for dna unwinding enzyme positioning and dna opening
Journal of Molecular Biology, 1997Co-Authors: Joseph A Farah, Gerald R SmithAbstract:Abstract The Escherichia coli RecBCD enzyme unwinds DNA from a free double-stranded DNA end to produce single-stranded DNA intermediates of homologous recombination. In the absence of ATP RecBCD binds to a free DNA end to form an Initiation Complex for DNA unwinding. We studied the structure of these Complexes formed with blunt-ended, 5′-extended, and 3′-extended DNA. Reactivity to the single-stranded DNA-specific reagents KMnO 4 and dimethyl sulfate indicated that RecBCD opened, in a Mg 2+ -dependent manner, the terminal five or six base-pairs in each substrate. Thymine residues located four to six nucleotides from the 5′ end were only partially reactive to KMnO 4 , suggesting that part of the 5′-terminated strand was partially shielded by the enzyme. DNase I footprinting indicated that the enzyme positions itself relative to the end of the longer of the two strands, although an exception was noted. These results imply flexibility in the ability of RecBCD to open the DNA and position itself for unwinding on DNA with different types of ends. They also imply conformational differences of RecBCD enzyme bound to different types of ends; these conformational differences may be related to those occurring during the unwinding cycle.
Elisabetta Viani Puglisi - One of the best experts on this subject based on the ideXlab platform.
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dynamic interplay of rna and protein in the human immunodeficiency virus 1 reverse transcription Initiation Complex
Journal of Molecular Biology, 2018Co-Authors: Aaron T. Coey, Kevin P. Larsen, Daniel J. Barrero, Joseph D. Puglisi, Junhong Choi, Elisabetta Viani PuglisiAbstract:The Initiation of reverse transcription in human immunodeficiency virus-1 is a key early step in the virus replication cycle. During this process, the viral enzyme reverse transcriptase (RT) copies the single-stranded viral RNA (vRNA) genome into double-stranded DNA using human tRNALys3 as a primer for Initiation. The tRNA primer and vRNA genome contain several complementary sequences that are important for regulating reverse transcription Initiation kinetics. Using single-molecule Forster resonance energy transfer spectroscopy, we demonstrate that the vRNA-tRNA Initiation Complex is conformationally heterogeneous and dynamic in the absence of RT. As shown previously, nucleic acid-RT interaction is characterized by rapid dissociation constants. We show that extension of the vRNA-tRNA primer binding site helix from 18 base pairs to 22 base pairs stabilizes RT binding to the Complex and that the tRNA 5' end has a role in modulating RT binding. RT occupancy on the Complex stabilizes helix 1 formation and reduces global structural heterogeneity. The stabilization of helix 1 upon RT binding may serve to destabilize helix 2, the first pause site for RT during Initiation, during later steps of reverse transcription Initiation.
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Architecture of an HIV-1 reverse transcriptase Initiation Complex.
Nature, 2018Co-Authors: Kevin P. Larsen, Yamuna Kalyani Mathiharan, Kalli Kappel, Aaron T. Coey, Dong-hua Chen, Daniel J. Barrero, Lauren Madigan, Joseph D. Puglisi, Georgios Skiniotis, Elisabetta Viani PuglisiAbstract:Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in viral infection1 and a common target of antiretroviral drugs2. The reaction is catalysed by viral reverse transcriptase (RT)3,4 that is packaged in an infectious virion with two copies of viral genomic RNA5 each bound to host lysine 3 transfer RNA (tRNALys3), which acts as a primer for Initiation of reverse transcription6,7. Upon viral entry into cells, Initiation is slow and non-processive compared to elongation8,9. Despite extensive efforts, the structural basis of RT function during Initiation has remained a mystery. Here we use cryo-electron microscopy to determine a three-dimensional structure of an HIV-1 RT Initiation Complex. In our structure, RT is in an inactive polymerase conformation with open fingers and thumb and with the nucleic acid primer–template Complex shifted away from the active site. The primer binding site (PBS) helix formed between tRNALys3 and HIV-1 RNA lies in the cleft of RT and is extended by additional pairing interactions. The 5′ end of the tRNA refolds and stacks on the PBS to create a long helical structure, while the remaining viral RNA forms two helical stems positioned above the RT active site, with a linker that connects these helices to the RNase H region of the PBS. Our results illustrate how RNA structure in the Initiation Complex alters RT conformation to decrease activity, highlighting a potential target for drug action. A cryo-EM structure of an Initiation Complex of HIV-1 reverse transcriptase sheds light on the Initiation of reverse transcription of viral RNA.