The Experts below are selected from a list of 255 Experts worldwide ranked by ideXlab platform
Roger D. Kornberg - One of the best experts on this subject based on the ideXlab platform.
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Eukaryotic RNA Polymerase II
Nucleic Acid Polymerases, 2013Co-Authors: David A. Bushnell, Roger D. KornbergAbstract:Structures of yeast RNA Polymerase II, alone and in the act of transcription with associated DNA and RNA, have been determined to near-atomic resolution. The structures illuminate the basis for the fidelity of transcription, for translocation on the DNA template, and for release of the product RNA. Structures of an RNA Polymerase II-general transcription factor complex have led to a model for a closed transcription initiation complex and have suggested a possible basis for promoter escape.
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RNA Polymerase II Transcription: Structure and Mechanism
Biochimica et biophysica acta, 2012Co-Authors: Xin Liu, David A. Bushnell, Roger D. KornbergAbstract:A minimal RNA Polymerase II (pol II) transcription system comprises the Polymerase and five general transcription factors (GTFs) TFIIB, -D, -E, -F, and -H. The addition of Mediator enables a response to regulatory factors. The GTFs are required for promoter recognition and the initiation of transcription. Following initiation, pol II alone is capable of RNA transcript elongation and of proofreading. Structural studies reviewed here reveal roles of GTFs in the initiation process and shed light on the transcription elongation mechanism. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.
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Quantitation of the RNA Polymerase II Transcription Machinery in Yeast
The Journal of biological chemistry, 2001Co-Authors: Tilman Borggrefe, Ralph E. Davis, Avital Bareket-samish, Roger D. KornbergAbstract:Abstract TAP tags and dot blot analysis have been used to measure the amounts of RNA Polymerase II transcription proteins in crude yeast extracts. The measurements showed comparable amounts of RNA Polymerase II, TFIIE, and TFIIF, lower levels of TBP and TFIIB, and still lower levels of Mediator and TFIIH. These findings are consistent with the presumed roles of the transcription proteins, but do not support the idea of their recruitment in a single large complex to RNA Polymerase II promoters. The approach employed here can be readily extended to quantitative analysis of the entire yeast proteome.
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A multiprotein complex that interacts with RNA Polymerase II elongator.
The Journal of biological chemistry, 2001Co-Authors: Yuichiro Takagi, Yiwei Jiang, Masao Tokunaga, Hediye Erdjument-bromage, Paul Tempst, Roger D. KornbergAbstract:Abstract A three-subunit Hap complex that interacts with the RNA Polymerase II Elongator was isolated from yeast. Deletions of genes for two Hap subunits, HAP1 andHAP3, confer pGKL killer-insensitive and weak Elongator phenotypes. Preferential interaction of the Hap complex with free rather than RNA Polymerase II-associated Elongator suggests a role in the regulation of Elongator activity.
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Conserved Structures of Mediator and RNA Polymerase II Holoenzyme
Science (New York N.Y.), 1999Co-Authors: Francisco J. Asturias, Claes M. Gustafsson, Lawrence C. Myers, Yiwei Jiang, Roger D. KornbergAbstract:Single particles of the mediator of transcriptional regulation (Mediator) and of RNA Polymerase II holoenzyme were revealed by electron microscopy and image processing. Mediator alone appeared compact, but at high pH or in the presence of RNA Polymerase II it displayed an extended conformation. Holoenzyme contained Mediator in a fully extended state, partially enveloping the globular Polymerase, with points of apparent contact in the vicinity of the Polymerase carboxyl-terminal domain and the DNA-binding channel. A similarity in appearance and conformational behavior of yeast and murine complexes indicates a conservation of Mediator structure among eukaryotes.
Danny Reinberg - One of the best experts on this subject based on the ideXlab platform.
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Human Elongator facilitates RNA Polymerase II transcription through chromatin
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Jae-hyun Kim, William S. Lane, Danny ReinbergAbstract:A human Elongator complex was purified from HeLa cells and found to be composed of three polypeptides. Human Elongator contains histone acetyltransferase activity with specificity to histone H3 and, to a much lesser extent, to histone H4. Although many reports have suggested a role for the yeast Elongator in transcription elongation through chromatin templates, no direct evidence supporting this function exists. In the present study, we demonstrate that the human Elongator facilitates transcription by RNA Polymerase II in a chromatin- and acetyl-CoA-dependent manner. The complex was found to directly interact with RNA Polymerase II but failed to interact with other factors that facilitated RNA Polymerase II to traverse through nucleosomes. From our results, we postulate that different mechanisms operate to ensure efficient transcription by RNA Polymerase II on chromatin templates.
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eLS - RNA Polymerase II Holoenzyme and Transcription Factors
Encyclopedia of Life Sciences, 2001Co-Authors: Michael Hampsey, Danny ReinbergAbstract:Transcription of protein-encoding genes requires a host of auxiliary factors that assemble with RNA Polymerase II on promoter DNA. Keywords: transcription; initiation; RNA Polymerase II; general transcription factor; core promoter; holoenzyme
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RNA Polymerase II as a control panel for multiple coactivator complexes
Current Opinion in Genetics & Development, 1999Co-Authors: Michael Hampsey, Danny ReinbergAbstract:1999 marks the 30th anniversary of the reported discovery of σ factor and the bacterial RNA Polymerase holoenzyme. In 1994, an RNA Polymerase II complex was discovered in yeast — mammalian complexes were subsequently identified. Recent developments regarding the composition and function of RNA Polymerase II complexes suggest, however, that the concept of the holoenzyme, as defined in bacteria, might not be relevant to eukaryotes.
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A Human RNA Polymerase II Complex Containing Factors That Modify Chromatin Structure
Molecular and cellular biology, 1998Co-Authors: Helen Cho, George Orphanides, Xiaoqing Sun, Xiang-jiao Yang, Vasily Ogryzko, Emma Lees, Yoshihiro Nakatani, Danny ReinbergAbstract:We have isolated a human RNA Polymerase II complex that contains chromatin structure remodeling activity and histone acetyltransferase activity. This complex contains the Srb proteins, the Swi-Snf complex, and the histone acetyltransferases CBP and PCAF in addition to RNA Polymerase II. Notably, the general transcription factors are absent from this complex. The complex was purified by two different methods: conventional chromatography and affinity chromatography using antibodies directed against CDK8, the human homolog of the yeast Srb10 protein. Protein interaction studies demonstrate a direct interaction between RNA Polymerase II and the histone acetyltransferases p300 and PCAF. Importantly, p300 interacts specifically with the nonphosphorylated, initiation-competent form of RNA Polymerase II. In contrast, PCAF interacts with the elongation-competent, phosphorylated form of RNA Polymerase II.
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Specific interaction between the nonphosphorylated form of RNA Polymerase II and the TATA-binding protein.
Cell, 1992Co-Authors: Anny Usheva, Danny Reinberg, Edio Maldonado, Anat Goldring, Christo Houbavi, Yosef AloniAbstract:Fractionation of a transcription-competent HeLa cell extract on a column containing one copy of the heptamer repeat (YSPTSPS) present in the carboxy-terminal domain (CTD) of the largest subunit of RNA Polymerase II resulted in the loss of transcriptional activity. Fractionation of the extracts on columns containing mutations of the heptamer repeat was without effect. Such transcriptionally inactive extracts regained their ability to specifically transcribe different class II promoters upon the addition of human TFIID, recombinant yeast TATA-binding protein (TBP), or proteins bound to the column. Fractionation of RNA Polymerase II on columns containing human or yeast TBP resulted in the specific retention of the nonphosphorylated form of RNA Polymerase II. The phosphorylated form of the enzyme was unable to interact with TBP. The specific interaction of RNA Polymerase II with TBP was mediated by the CTD of RNA Polymerase II.
Richard A Young - One of the best experts on this subject based on the ideXlab platform.
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A mammalian SRB protein associated with an RNA Polymerase II holoenzyme
Nature, 1996Co-Authors: David M. Chao, Ellen L. Gadbois, Peter J. Murray, Stephen Anderson, Michelle S. Sonu, Jeffrey D. Parvin, Richard A YoungAbstract:A LARGE multisubunit complex containing RNA Polymerase II, general transcription factors and SRB regulatory proteins initiates transcription of class II genes in yeast cells1–4. The SRB proteins are a hallmark of this RNA Polymerase II holoenzyme as they are found only in this complex, where they contribute to the response to regulators4–8. We have now isolated a human honiologue of the yeast SRB7 gene and used antibodies against human SRB7 protein to purify and characterize a mammalian RNA Polymerase II holoenzyme containing the general transcription factors TFIIE and TFIIH. This holoenzyme is more responsive to transcriptional activators than core RNA Polymerase II when assayed in the presence of coactivators.
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RNA Polymerase II Holoenzyme Contains SWI/SNF Regulators Involved in Chromatin Remodeling
Cell, 1996Co-Authors: Christine D. Wilson, David M. Chao, Anthony N. Imbalzano, Gavin R. Schnitzler, Robert E. Kingston, Richard A YoungAbstract:The RNA Polymerase II holoenzyme contains RNA Polymerase II, a subset of general transcription factors and SRB regulatory proteins. We report here that SWI and SNF gene products, previously identified as global gene regulators whose functions include remodeling chromatin, are also integral components of the yeast RNA Polymerase II holoenzyme. The SWI/SNF proteins are components of the SRB complex, also known as the mediator, which is tightly associated with the RNA Polymerase II C-terminal repeat domain. The SWI/SNF components provide the holoenzyme with the capacity to disrupt nucleosomal DNA and thus facilitate stable binding of various components of the transcription initiation complex at promoters.
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Purification of yeast RNA Polymerase II holoenzymes.
Methods in enzymology, 1996Co-Authors: Anthony J. Koleske, David M. Chao, Richard A YoungAbstract:Publisher Summary This chapter describes methods for the purification and assay of the two largest forms of RNA Polymerase II holoenzyme from the yeast Saccharomyces cerevisiae . Use of the purified holoenzyme for transcription studies in vitro should yield new and exciting clues regarding the mechanisms underlying transcriptional regulation. RNA Polymerase II can be purified from yeast in a high molecular weight complex called an RNA Polymerase II holoenzyme. This form of RNA Polymerase II appears to be responsible for transcription initiation in vivo . All forms of RNA Polymerase II holoenzyme described thus far lack the general transcription factors TBP and TFIIE (transcription factor IIE), and these factors must be added to obtain transcription in vitro. TFIIB is also required for transcription by some holoenzyme preparations. Holoenzyme and factor preparations should be dialyzed against a low-salt buffer, such as buffer F, to reduce the salt content prior to assay. AlteRNAtively, the salt concentration can be determined by conductivity measurements, and the salt concentration in the transcription assay buffer can be adjusted accordingly.
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General requirement for RNA Polymerase II holoenzymes in vivo
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Craig.m Thompson, Richard A YoungAbstract:Abstract Yeast RNA Polymerase II holoenzymes have been described that consist of RNA Polymerase II, a subset of general transcription factors, and nine SRB regulatory proteins. The feature that distinguishes the RNA Polymerase II holoenzymes from other forms of RNA Polymerase II in the cell is their tight association with SRB proteins. We investigated the fraction of genes that require SRB proteins in vivo by examining the effect of temperature-sensitive mutations in SRB genes on transcription by RNA Polymerase II. Upon transfer to the restrictive temperature, there is a rapid and general shutdown of mRNA synthesis in srb mutant cells. These data, combined with the observation that essentially all of the SRB protein in cells is tightly associated with RNA Polymerase II molecules, argue that SRB-containing holoenzymes are the form of RNA Polymerase II recruited to most promoters in the cell.
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Association of an activator with an RNA Polymerase II holoenzyme.
Genes & development, 1995Co-Authors: Christoph J. Hengartner, Anthony J. Koleske, Craig.m Thompson, David M. Chao, Sha-mei Liao, Jianhua Zhang, Sara Okamura, Richard A YoungAbstract:RNA Polymerase II holoenzymes have been described that consist of RNA Polymerase II, a subset of general transcription factors, and four SRB proteins. The SRB proteins, which were identified through a selection for genes involved in transcription initiation by RNA Polymerase II in vivo, are a hallmark of the holoenzyme. We report here the isolation and characterization of additional SRB genes. We show that the products of all nine SRB genes identified thus far are components of the RNA Polymerase II holoenzyme and are associated with a holoenzyme subcomplex termed the mediator of activation. The holoenzyme is capable of responding to a transcriptional activator, suggesting a model in which activators function, in part, through direct interactions with the holoenzyme. Immunoprecipitation experiments with anti-SRB5 antibodies demonstrate that the acidic activating domain of VP16 specifically binds to the holoenzyme. Furthermore, the holoenzyme and the mediator subcomplex bind to a VP16 affinity column. These results provide a more complete description of the RNA Polymerase II holoenzyme and suggest that this form of the transcription apparatus can be recruited to promoters via direct interactions with activators.
R C Conaway - One of the best experts on this subject based on the ideXlab platform.
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The RNA Polymerase II elongation complex
Annual Review of Biochemistry, 2003Co-Authors: Ali Shilatifard, R C Conaway, J W ConawayAbstract:▪ Abstract Synthesis of eukaryotic mRNA by RNA Polymerase II is an elaborate biochemical process that requires the concerted action of a large set of transcription factors. RNA Polymerase II transcription proceeds through multiple stages designated preinitiation, initiation, and elongation. Historically, studies of the elongation stage of eukaryotic mRNA synthesis have lagged behind studies of the preinitiation and initiation stages; however, in recent years, efforts to elucidate the mechanisms governing elongation have led to the discovery of a diverse collection of transcription factors that directly regulate the activity of elongating RNA Polymerase II. Moreover, these studies have revealed unanticipated roles for the RNA Polymerase II elongation complex in such processes as DNA repair and recombination and the proper processing and nucleocytoplasmic transport of mRNA. Below we describe these recent advances, which highlight the important role of the RNA Polymerase II elongation complex in regulation o...
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The RNA Polymerase II elongation complex
Annual review of biochemistry, 2003Co-Authors: Ali Shilatifard, R C Conaway, J W ConawayAbstract:Synthesis of eukaryotic mRNA by RNA Polymerase II is an elaborate biochemical process that requires the concerted action of a large set of transcription factors. RNA Polymerase II transcription proceeds through multiple stages designated preinitiation, initiation, and elongation. Historically, studies of the elongation stage of eukaryotic mRNA synthesis have lagged behind studies of the preinitiation and initiation stages; however, in recent years, efforts to elucidate the mechanisms governing elongation have led to the discovery of a diverse collection of transcription factors that directly regulate the activity of elongating RNA Polymerase II. Moreover, these studies have revealed unanticipated roles for the RNA Polymerase II elongation complex in such processes as DNA repair and recombination and the proper processing and nucleocytoplasmic transport of mRNA. Below we describe these recent advances, which highlight the important role of the RNA Polymerase II elongation complex in regulation of eukaryotic gene expression.
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Preparation and assay of RNA Polymerase II elongation factors elongin and ELL.
Methods in enzymology, 2003Co-Authors: Stephanie E. Kong, Ali Shilatifard, R C Conaway, J W ConawayAbstract:Publisher Summary This chapter describes methods for preparing recombinant Elongin and ELL, and for assaying their activities in promoting rapid and efficient elongation by RNA Polymerase II. Based on varied evidence from biochemical studies, Elongin and ELL appear to function similarly to increase the overall rate of transcript elongation by RNA Polymerase II by a mechanism that involves suppression of transient pausing by Polymerase at many sites along the DNA template. Both Elongin and ELL appear to suppress RNA Polymerase II, pausing through direct interactions with Polymerase by decreasing the time the enzyme spends in inactive conformations. Elongin and ELL can both stimulate the rate of elongation by purified RNA Polymerase II in vitro. Two approaches for assaying Elongin and ELL transcription activities have proven successful. First, Elongin and ELL have both been shown to be capable of stimulating the rate of elongation by RNA Polymerase II that has initiated transcription from a promoter in the presence of the general initiation factors TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Second, Elongin and ELL have both been shown to be capable of stimulating the rate of elongation by RNA Polymerase II on oligo (dC)-tailed DNA templates in the absence of auxillary transcription factors.
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Assays for Investigating the Mechanism of Promoter Escape by RNA Polymerase II
Methods in enzymology, 2003Co-Authors: Arik Dvir, J W Conaway, R C ConawayAbstract:Publisher Summary This chapter describes the methods and approaches that have proven useful in investigations of the mechanism of promoter escape by RNA Polymerase II, and should be valuable for future efforts to define the mechanism of important transcriptional stage in detail. It discusses that transcription initiation by RNA Polymerase II from its promoters is a complex process that requires at minimum the five general initiation factors TFIIB, TBP, TFIIE, TFIIF, and TFIIH and an ATP (dATP) cofactor. Biochemical studies of transcription initiation by RNA Polymerase II, in this minimal enzyme system, has revealed that initiation occurs by a multistep mechanism begins with the assembly of Polymerase. All five general initiation factor in a stable preinitiation complex at the promoter and proceeds with ATP(dATP)-dependent unwinding of promoter DNA surrounding the transcriptional start site by the TFIIH XPB DNA helicase to form the open complex, synthesis of the first few phosphodiester bonds of nascent transcripts, and escape of Polymerase from the promoter. Thus, efficient promoter escape by RNA Polymerase II requires that the early elongation complex undergo a critical ATP (dATP)-dependent structural transition that most likely depends on the interaction of Polymerase.
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Control of elongation by RNA Polymerase II.
Trends in biochemical sciences, 2000Co-Authors: J W Conaway, Ali Shilatifard, Arik Dvir, R C ConawayAbstract:The elongation stage of eukaryotic mRNA synthesis can be regulated by transcription factors that interact directly with the RNA Polymerase II (pol II) elongation complex and by activities that modulate the structure of its chromatin template. Recent studies have revealed new elongation factors and have implicated the general initiation factors TFIIE, TFIIF and TFIIH, as well as the C-terminal domain (CTD) of the largest subunit of pol II, in elongation. The recently reported high-resolution crystal structure of RNA Polymerase II, which provides insight into the architecture of the elongation complex, marks a new era of investigation into transcription elongation.
J W Conaway - One of the best experts on this subject based on the ideXlab platform.
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The RNA Polymerase II elongation complex
Annual Review of Biochemistry, 2003Co-Authors: Ali Shilatifard, R C Conaway, J W ConawayAbstract:▪ Abstract Synthesis of eukaryotic mRNA by RNA Polymerase II is an elaborate biochemical process that requires the concerted action of a large set of transcription factors. RNA Polymerase II transcription proceeds through multiple stages designated preinitiation, initiation, and elongation. Historically, studies of the elongation stage of eukaryotic mRNA synthesis have lagged behind studies of the preinitiation and initiation stages; however, in recent years, efforts to elucidate the mechanisms governing elongation have led to the discovery of a diverse collection of transcription factors that directly regulate the activity of elongating RNA Polymerase II. Moreover, these studies have revealed unanticipated roles for the RNA Polymerase II elongation complex in such processes as DNA repair and recombination and the proper processing and nucleocytoplasmic transport of mRNA. Below we describe these recent advances, which highlight the important role of the RNA Polymerase II elongation complex in regulation o...
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The RNA Polymerase II elongation complex
Annual review of biochemistry, 2003Co-Authors: Ali Shilatifard, R C Conaway, J W ConawayAbstract:Synthesis of eukaryotic mRNA by RNA Polymerase II is an elaborate biochemical process that requires the concerted action of a large set of transcription factors. RNA Polymerase II transcription proceeds through multiple stages designated preinitiation, initiation, and elongation. Historically, studies of the elongation stage of eukaryotic mRNA synthesis have lagged behind studies of the preinitiation and initiation stages; however, in recent years, efforts to elucidate the mechanisms governing elongation have led to the discovery of a diverse collection of transcription factors that directly regulate the activity of elongating RNA Polymerase II. Moreover, these studies have revealed unanticipated roles for the RNA Polymerase II elongation complex in such processes as DNA repair and recombination and the proper processing and nucleocytoplasmic transport of mRNA. Below we describe these recent advances, which highlight the important role of the RNA Polymerase II elongation complex in regulation of eukaryotic gene expression.
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Preparation and assay of RNA Polymerase II elongation factors elongin and ELL.
Methods in enzymology, 2003Co-Authors: Stephanie E. Kong, Ali Shilatifard, R C Conaway, J W ConawayAbstract:Publisher Summary This chapter describes methods for preparing recombinant Elongin and ELL, and for assaying their activities in promoting rapid and efficient elongation by RNA Polymerase II. Based on varied evidence from biochemical studies, Elongin and ELL appear to function similarly to increase the overall rate of transcript elongation by RNA Polymerase II by a mechanism that involves suppression of transient pausing by Polymerase at many sites along the DNA template. Both Elongin and ELL appear to suppress RNA Polymerase II, pausing through direct interactions with Polymerase by decreasing the time the enzyme spends in inactive conformations. Elongin and ELL can both stimulate the rate of elongation by purified RNA Polymerase II in vitro. Two approaches for assaying Elongin and ELL transcription activities have proven successful. First, Elongin and ELL have both been shown to be capable of stimulating the rate of elongation by RNA Polymerase II that has initiated transcription from a promoter in the presence of the general initiation factors TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Second, Elongin and ELL have both been shown to be capable of stimulating the rate of elongation by RNA Polymerase II on oligo (dC)-tailed DNA templates in the absence of auxillary transcription factors.
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Assays for Investigating the Mechanism of Promoter Escape by RNA Polymerase II
Methods in enzymology, 2003Co-Authors: Arik Dvir, J W Conaway, R C ConawayAbstract:Publisher Summary This chapter describes the methods and approaches that have proven useful in investigations of the mechanism of promoter escape by RNA Polymerase II, and should be valuable for future efforts to define the mechanism of important transcriptional stage in detail. It discusses that transcription initiation by RNA Polymerase II from its promoters is a complex process that requires at minimum the five general initiation factors TFIIB, TBP, TFIIE, TFIIF, and TFIIH and an ATP (dATP) cofactor. Biochemical studies of transcription initiation by RNA Polymerase II, in this minimal enzyme system, has revealed that initiation occurs by a multistep mechanism begins with the assembly of Polymerase. All five general initiation factor in a stable preinitiation complex at the promoter and proceeds with ATP(dATP)-dependent unwinding of promoter DNA surrounding the transcriptional start site by the TFIIH XPB DNA helicase to form the open complex, synthesis of the first few phosphodiester bonds of nascent transcripts, and escape of Polymerase from the promoter. Thus, efficient promoter escape by RNA Polymerase II requires that the early elongation complex undergo a critical ATP (dATP)-dependent structural transition that most likely depends on the interaction of Polymerase.
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Control of elongation by RNA Polymerase II.
Trends in biochemical sciences, 2000Co-Authors: J W Conaway, Ali Shilatifard, Arik Dvir, R C ConawayAbstract:The elongation stage of eukaryotic mRNA synthesis can be regulated by transcription factors that interact directly with the RNA Polymerase II (pol II) elongation complex and by activities that modulate the structure of its chromatin template. Recent studies have revealed new elongation factors and have implicated the general initiation factors TFIIE, TFIIF and TFIIH, as well as the C-terminal domain (CTD) of the largest subunit of pol II, in elongation. The recently reported high-resolution crystal structure of RNA Polymerase II, which provides insight into the architecture of the elongation complex, marks a new era of investigation into transcription elongation.
