Steven M Hahn
Fred Hutchinson Cancer Research Center
Project start date: 2005-09-01
Project end date: 2014-01-31
Sponsored Links Excellgen http://Excellgen.com
Mechanisms Of Eukaryotic Transcription Activation
Steven M Hahn, Member
Fred Hutchinson Cancer Research Center
box 19024, 1100 Fairview Ave N
seattle, Wa 981091024
Grant 5R01GM075114-04 from National Institute Of General Medical Sciences IRG: MGA
Abstract: Activation of transcription is the ultimate endpoint for many signal transaction and developmental pathways, and understanding the mechanism of activation is a key to understanding the action of these pathways. From previous studies, it is clear that disruption of normal gene regulation by mutations in gene-specific transcription activators can lead to cancer and other diseases. The broad long-term objectives of this proposal are to determine the mechanisms used by gene-specific transcription factors to activate transcription by RNA Polymerase II (Pol II). The proposed work will provide a basis for understanding gene regulation in normal and diseased states at the molecular level. The specific aims of this work will utilize biochemical, structural, and molecular genetic methods to examine the direct targets of several transcription activators and the mechanism whereby contact with these targets stimulates transcription. Using a newly developed method for mapping protein-protein contacts within large complexes, we will identify direct targets of activation domains in several model activators in the presence or absence of chromatin. Biochemical and molecular genetic studies will be conducted to test the relevance of these interactions in transcription. We will extend these studies to examine the targets of a different activator class acting at TATA-less promoters. After mapping these activator-target interactions by hydroxyl radical cleavage and direct protein-protein interaction assays, we will determine the structure of the activation domains in combination with their relevant targets. Finally, by blocking assembly of the Preinitiation Complex at intermediate stages, we will examine the mechanism by which activator contact with one of these targets, the SAGA coactivator complex, stimulates transcription. Our proposed work will illuminate important mechanisms and principles of transcriptional regulation
Project start date: 2005-09-01
Project end date: 2009-08-31
5R01GM075114-04 (2008): $475349
5R01GM075114-03 (2007): $475477
5R01GM075114-02 (2006): $475624
Grants awarded to Steven M Hahn
Molecular Analysis Of Eukaryotic Transcription
Steven M Hahn, Member
Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 981091024
Grant 5R01GM053451-11 from National Institute Of General Medical Sciences IRG: MGB
Abstract: Transcription by eukaryotic RNA Polymerases (Pols) is the ultimate target for many signal transduction and developmental pathways, and understanding the mechanism of transcription is a key to understanding the mechanism of gene regulation. From previous studies, it is clear that disruption of normal gene regulation by mutations in gene-specific transcription factors, chromatin modifying factors, or general transcription factors can lead to cancer and other diseases. The broad long-term objectives of this proposal are to determine the mechanisms utilized by the transcription machinery to promote transcription by Pol II and Pol III. The proposed work will provide a basis for understanding gene regulation in normal and diseased states at the molecular level. The specific aims of this work will utilize biochemical, molecular, and genetic methods to examine the structures and mechanisms of the transcription machinery directing RNA Pols II and III in S. cerevisiae. Using a newly developed method for mapping the structural arrangement of large complexes, the structure of the Pol II pre-initiation Complex and Open Complex will be determined. The structures of these complexes will be compared on different promoter types. Based on these results, models for the mechanism of the general factors in transcription initiation will be derived and these models will be tested using biochemical and genetic assays. Similar methods will be utilized to determine the arrangement of the Pol III general factor Brf1 within the Pol III transcription machinery, permitting comparison of conserved and non conserved mechanisms utilized by different nuclear Pols. Finally, the mechanism of two cyclin-dependent kinases that regulate transcription during elongation will be explored. Analog-sensitive versions of these kinases will be utilized to identify relevant kinase targets. In vitro and in vivo assays will be used to determine the mechanisms whereby kinase activity regulates transcription.
Keywords: DNA directed RNA polymerase, fungal genetics, genetic regulation, genetic transcription, transcription factor, chromatin, crosslink, cyclin dependent kinase, gene mutation, genetic promoter element, phosphorylation, physical model, Saccharomyces cerevisiae, antiserum, laboratory rabbit
Project start date: 1995-09-30
Project end date: 2009-03-31
5R01GM053451-11 (2006): $665161
2R01GM053451-10 (2005): $570007
5R01GM053451-15 (2010): $653721
2R01GM053451-14 (2009): $641244
MOLECULAR ANALYSIS OF RNAPII TRANSCRIPTION INITIATION
Steven M Hahn, Member
Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 981091024
Grant 5R01GM042551-09 from National Institute Of General Medical Sciences IRG: MBY
Abstract: The broad, long-term objectives of this proposal are to determine at a molecular level, the mechanisms of the Pol II general transcription factors and how their activity can be regulated. This study is important because understanding of the Pol II transcription machinery is essential to understanding how gene-specific regulatory factors function. This is because the transcription machinery is the ultimate target of gene- specific regulatory factors. Understanding the fundamental process of gene control will provide a basis for understanding many types of diseases such as cancer which often result from aberrant regulation of transcription initiation. In addition, some diseases involving defects in DNA repair are caused by mutations in the Pol II general factors. The specific aims of this project are to (i) study through molecular, genetic, and biochemical methods, the function of TFIIA, a regulatory general transcription factor. We will isolate suppressors of conditional TFIIA mutations, continue a structure-function analysis of TFIIA function, and characterize at least two inhibitors of Pol II transcription whose inhibitory activity is blocked by TFIIA. (ii) We will continue our purification of the Pol II general transcription machinery from yeast so we can use these factors in our study of general factor function. (iii) we will use yeast genetics to isolate and characterize activator independent mutations in TBP (the TATA binding protein) and/or TFIIB to study the mechanism of gene regulation. These mutations will allow us to test two specific models for how acidic transcriptional activators act. Our work will involve molecular techniques such as gene cloning, mutagenesis and PCR, biochemical fractionation, in vitro transcription, and protein chemistry, as well as yeast molecular genetics.
Keywords: DNA directed RNA polymerase, genetic regulation, genetic transcription, molecular genetics, transcription factor, DNA binding protein, crosslink, fungal genetics, genetic promoter element, protein sequence, suppressor mutation, affinity chromatography, antiserum, laboratory mouse, laboratory rabbit, molecular cloning, mutant, polymerase chain reaction, site directed mutagenesis
Project start date: 1989-07-01
Project end date: 1999-06-30
5R01GM042551-09 (1997): $300580
5R01GM042551-07 (1995): $302737
Molecular Analysis Of RNAP II And III Transcription
Steven M Hahn, Member
Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 981091024
Grant 5R01GM053451-09 from National Institute Of General Medical Sciences IRG: CDF
Abstract: The broad, long-term objectives of this proposal are to determine at a molecular level, the mechanism of transcription initiation by RNA Polymerases II and III. These studies will lead to a better understanding of the transcription machinery and how it is regulated. This is important because the transcription machinery is the ultimate target of many signal transduction, cell cycle, and developmental pathways. Understanding the fundamental process of gene control will provide a basis for understanding many types of diseases such as cancer and syndromes involving DNA repair defects which can result from aberrant gene regulation, or from mutations in the transcription machinery. The specific aims of this project are designed to study through molecular, biochemical and genetic methods, the mechanisms of transcription initiation and reinitiation utilized by RNA Polymerases II and III. The role of novel factors, which interact with the TFIIA terminal domain in preinitiation complexes (PICs), will be identified. The mechanism of Pol II transcription reinitiation will be investigated with a focus on the role of the mediator factor and identification of a form of Pol II used in the reinitiation reaction. The role of several distinct mediator complexes in gene regulation will be studied. The role of the core domain of the Pol III factor BRF will be elucidated, and compared to the role of the homologous region of the Pol II factor TFIIB. Finally, the target of the zinc ribbon domains in TFIIB and Brf will be identified, comparing the role of these domains in Pol II and III transcription.
Keywords: DNA directed RNA polymerase, enzyme mechanism, genetic transcription, transcription factor, DNA binding protein, chemical association, enzyme activity, fungal genetics, genetic regulation, mutant, protein structure function, crosslink, laboratory mouse, laboratory rabbit, polymerase chain reaction, site directed mutagenesis
Project start date: 1995-09-30
Project end date: 2005-03-31
5R01GM053451-09 (2004): $269048
5R01GM053451-08 (2003): $269679
5R01GM053451-07 (2002): $270286
2R01GM053451-06 (2001): $270868
Sponsored Links Excellgen http://Excellgen.com
MOLECULAR ANALYSIS OF RNAPIII TRANSCRIPTION INITIATION
Steven M Hahn, Member
Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 981091024
Grant 5R01GM053451-05 from National Institute Of General Medical Sciences IRG: MBY
Abstract: The broad, long-term objectives of this proposal are to determine at a molecular level the mechanism of transcription initiation by RNA polymerase III and how this relates to the initiation mechanisms for the other nuclear polymerases. This will lead to a better understanding of the transcription initiation machinery and how it is regulated. This is important because the transcription machinery is the ultimate target of many signal transduction pathways and gene-specific regulatory factors. Understanding the fundamental process of gene control will provide a basis for understanding many types of diseases such as cancer, which often result from aberrant regulation of transcription initiation. In addition, other diseases resulting from defects in repair of DNA damage are caused by mutations in components of the transcription machinery. The specific aims of this project are designed to study through molecular, genetic and biochemical methods, the function of TFIIIB, a multi-subunit component of the Pol III transcription machinery. Dr. Hahn will complete the cloning of the TFIIIB subunit TFIIIB90 and perform a molecular and genetic analysis of its function. Dr. Hahn will continue the genetic and biochemical analysis of BRF1, a second subunit of TFIIIB. He will also use a genetic approach to understand the role of TATA binding protein, a third subunit of TFIIIB, in Pol III transcription Finally, Dr. Hahn has devised a strategy to examine in detail the role of the individual TFIIIB subunits in the process of polymerase recruitment and open complex formation. His work will involve molecular techniques (gene cloning, mutagenesis, and PCR), biochemical fractionation, in vitro transcription, protein biochemistry, and yeast molecular genetics.
Keywords: DNA directed RNA polymerase, genetic transcription, transcription factor, DNA binding protein, fungal genetics, mutant, protein structure /function, molecular cloning, polymerase chain reaction, site directed mutagenesis
Project start date: 1995-09-30
Project end date: 2001-03-31
5R01GM053451-05 (1999): $265730
5R01GM053451-04 (1998): $251320
5R01GM053451-03 (1997): $247945
1R01GM053451-01 (1995): $124259
MOLECULAR ANALYSIS OF RNAPII TRANSCRIPTION INITIATION
Steven M Hahn, Member
Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 981091024
Grant 5R01GM042551-05 from National Institute Of General Medical Sciences IRG: MBY
Abstract: The broad long-term objectives of this proposal are to (10 elucidate at a molecular level, the pathway of transcription initiation by RNA polymerase II (RNAIPII) and (2) determine how the steps in this pathway are affected by gene-specific regulatory proteins. The specific aims are to identify and purify the RNAPII general transcription factors for S. cerevisiae, to clone and analyze the gene encoding the TATA binding factor. TFIID, and to analyze at a biochemical level how this protein interacts with DNA as well as other components of the transcriptional machinery and gene-specific regulatory proteins. Understanding the fundamental process of gene control will provide a basis for understanding complex patterns of gene expression regulating development and the growth of animal cells. The yeast general transcription factors TFIIB,E will be identified by reconstitution of a homologous yeast in vitro transcription system. These factors, as well as the yeast general factor TFIIA will be purified. Using the already determined amino acid sequence of TFIID, the gene encoding this factor will be cloned and characterized. The yeast TFIID gene will be used to create TRIIF mutations in yeast to study its function, determine whether it is part of a multigene family and to study the regulation of its expression. Genes encoding factors which directly interact with TFIID will be identified by genetic screening and biochemical analysis. The TFIID gene will be mutagenized in vitro and the effect of these mutations will be assessed both in vivo and in vitro to define the functional domains of the protein. Using chemical premodification and mutagenesis of the TFIID DNA binding sites, the critical protein-DNA interactions will be determined. Finally, the assembly of the general factors with RNAPII will be studied in vitro using a native gel assay and DNAsel protection. The kinetics of general factor assembly will be examined in both the presence and absence of transcriptional regulatory proteins to identify regulated steps in the pathway of transcription initiation.
Keywords: RNA polymerase, nucleic acid sequence, regulatory gene, translation factor, DNA binding protein, Saccharomyces cerevisiae, fungal genetics, genetic mapping, genetic promoter element, protein sequence, immunochemistry, laboratory rabbit, molecular cloning, mutant, protein reconstitution, tissue /cell culture
Project start date: 1989-07-01
Project end date: 1994-06-30
5R01GM042551-05 (1993): $189349
5R01GM042551-04 (1992): $176772
MECHANISMS OF EUKARYOTIC TRANSCRIPTION ACTIVATION
Steven M Hahn
Fred Hutchinson Cancer Research Center, Box 19024, 1100 Fairview Ave N, Seattle, Wa 98109-1024
Grant 2R01GM075114-05A1 from National Institute Of General Medical Sciences
Abstract: Summary Activation of transcription is the ultimate endpoint for many signal transduction and developmental pathways, and understanding the mechanism of activation is a key to understanding gene regulation. From previous studies, it is clear that disruption of normal gene regulation by mutations in gene- specific transcription activators and coactivators can lead to cancer and other diseases. The broad long- term objectives of this proposal are to determine the mechanisms used by gene-specific activators and coactivators to regulate RNA polymerase II transcription. The proposed work will provide a basis for understanding gene regulation in normal and diseased states at the molecular level. The specific aims of this work utilize biochemical, structural, and molecular genetic methods to examine the direct targets of two activation domains and two coactivators. We will examine the structure of several acidic activator-coactivator complexes to understand how activators specifically recognize their targets, common principals of activator-target recognition and, more generally, the function of intrinsically disordered proteins. We will examine the interaction of the SAGA coactivator with TBP (TATA binding protein) and how this interaction is regulated by acetylation. Using a unique set of crosslinking reagents, we will examine the direct targets of the Mediator coactivator complex within the transcription machinery. In all cases, we will use yeast molecular genetics to test the functional significance of our biochemical results. Combined, our results will lead to a molecular model for how activators recognize their coactivator targets and how coactivators stimulate gene expression by direct interaction with the transcription machinery. The objective of this research is to understand the mechanism and regulation of transcription, the process of mRNA synthesis. Regulation of transcription is one of the key steps in control of cell growth, differentiation, and development, and defects in transcription directly contribute to many human illnesses. Understanding the mechanism of transcription and its regulation will form the basis for understanding the molecular defects in transcription disorders leading to many types of cancer, as well as heart disease, neurological disorders, and birth defects
Keywords: Acetylation; Affinity; Assay; Basal Transcription Factor; Bifunctional Reagents; Binding; Binding (Molecular Function); Bioassay; Biochemical; Biochemical Genetics; Biologic Assays; Biological; Biological Assay; Birth Defects; Cancers; Cardiac Diseases; Cardiac Disorders; Cell Communication and Signaling; Cell Signaling; Cellular Expansion; Cellular Growth; Code; Coding System; Complement; Complement Proteins; Complex; Congenital Abnormality; Congenital Anatomic Abnormality; Congenital Anatomical Abnormality; Congenital Defects; Congenital Deformity; Congenital Malformation; Cross-Linking Reagents; Crosslinking Reagents; DNA Polymerase II; DNA Polymerase epsilon; DNA-Dependent DNA Polymerase II; DNA-Dependent RNA Polymerase II; Defect; Development; Disease; Disorder; Eukaryota; Eukaryote; GTF2D; GTF2D1; Gene Action Regulation; Gene Expression; Gene Expression Regulation; Gene Products, RNA; Gene Regulation; Gene Regulation Process; Gene Transcription; General Transcription Factors; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic Transcription; Genetic defect; Genetic, Biochemical; Heart Diseases; Human; Human, General; Intracellular Communication and Signaling; Laboratories; Lead; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Maps; Mediator; Mediator of Activation; Mediator of activation protein; Messenger RNA; Methods; Modeling; Models, Molecular; Molecular; Molecular Genetic; Molecular Genetic Abnormality; Molecular Genetics; Molecular Interaction; Molecular Models; Mutation; Nervous System Diseases; Neurologic Disorders; Neurological Disorders; Nucleic Acid Biochemistry, Molecular Modeling; Pathway interactions; Pb element; Play; Pol II; Property; Property, LOINC Axis 2; Protein Binding; Protein/Amino Acid Biochemistry, Molecular Modeling; Proteins; RNA; RNA Expression; RNA Polymerase B; RNA Polymerase II; RNA Polymerase II TATA-Binding Protein; RNA, Messenger; RNA, Non-Polyadenylated; Reagent; Regulation; Research; Ribonucleic Acid; Role; SAGA; SCA17; SPT/ADA/Gcn5 Acetyltransferase; Signal Transduction; Signal Transduction Systems; Signaling; Structure; System; System, LOINC Axis 4; TATA Sequence-Binding Protein; TATA-Binding Protein; TATA-Box Binding Protein; TATA-Box Factor; TBP; TFIIE; Testing; Transcription; Transcription Activation; Transcription Activator; Transcription Coactivator; Transcription Factor Coactivator; Transcription Factor TBP; Transcription Factors, General; Transcription Process; Transcription Regulation; Transcription, Genetic; Transcriptional Activation; Transcriptional Activator; Transcriptional Activator/Coactivator; Transcriptional Coactivator; Transcriptional Control; Transcriptional Regulation; Up-Regulation; Work; Yeasts; base; biological signal transduction; cancer type; cell growth; disease/disorder; eukaryotida; gene product; genome mutation; heart disorder; heavy metal Pb; heavy metal lead; insight; mRNA; malignancy; molecular modeling; neoplasm/cancer; nervous system disorder; neurological disease; pathway; protein complex; protein protein interaction; public health relevance; social role; transcription factor IIE; transcription factor TFIIE; yeast genetics
Relevance: The objective of this research is to understand the mechanism and regulation of transcription, the process of mRNA synthesis. Regulation of transcription is one of the key steps in control of cell growth, differentiation, and development, and defects in transcription directly contribute to many human illnesses. Understanding the mechanism of transcription and its regulation will form the basis for understanding the molecular defects in transcription disorders leading to many types of cancer, as well as heart disease, neurological disorders, and birth defects
Project start date: 2005-09-01
Project end date: 2014-01-31
Budget start date: 19-MAR-2010
Budget end date: 31-JAN-2011
PFA/PA: PA-07-070
2R01GM075114-05A1 (2010): $520478
Transcriptional Regulation During Cell Growth Differentiation And Development
Steven M Hahn, Member
Federation Of Amer Soc For Exper Biology 9650 Rockville Pike Bethesda, Md 208143998
Grant 1R13HD054289-01 from National Institute Of Child Health And Human Development IRG: CHHD
Abstract: Partial funding is requested for a five day FASEB conference on "Transcriptional Regulation during Cell Growth, Differentiation, and Development" to be held at Saxtons River, Vermont in August 2006. Regulation of transcription is the ultimate and crucial endpoint for many signal transduction and developmental pathways that control cell growth, differentiation, organogenesis, and cancer. Understanding the networks and mechanisms that regulate transcription is necessary to understand normal growth and development, and transcriptional misregulation is the basis for many diseases, developmental defects, and neoplastic transformation. The purpose of this conference is to bring together biochemists and molecular biologists studying fundamental mechanisms of transcription, cell and developmental biologists studying biological processes dependent on transcriptional regulation, and scientists using global approaches to understand networks of interacting genes involved in transcriptional regulation of cell growth, differentiation, and disease models. This FASEB meeting is unique within these fields because rather than focusing on a single topic, it brings together scientists using different approaches to study transcriptional regulation in diverse systems. This interdisciplinary approach fosters new ways of thinking about problems, finding solutions to longstanding questions in the field, and, perhaps most importantly, cross-fertilization of basic science and biomedical research. The conference will also provide an up to date view of one of the most rapidly moving fields in biology.
Keywords: cell differentiation, cell growth regulation, genetic regulation, genetic transcription, meeting /conference /symposium, gene interaction, growth /development, travel
Project start date: 2006-08-05
Project end date: 2007-07-31
1R13HD054289-01 (2006): $6000
Sponsored Links Excellgen http://Excellgen.com
Mechanisms Of Eukaryotic Transcription Activation
Steven M Hahn, Member
Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 981091024
Grant 1R01GM075114-01 from National Institute Of General Medical Sciences IRG: MGA
Abstract: Activation of transcription is the ultimate endpoint for many signal transaction and developmental pathways, and understanding the mechanism of activation is a key to understanding the action of these pathways. From previous studies, it is clear that disruption of normal gene regulation by mutations in gene-specific transcription activators can lead to cancer and other diseases. The broad long-term objectives of this proposal are to determine the mechanisms used by gene-specific transcription factors to activate transcription by RNA Polymerase II (Pol II). The proposed work will provide a basis for understanding gene regulation in normal and diseased states at the molecular level. The specific aims of this work will utilize biochemical, structural, and molecular genetic methods to examine the direct targets of several transcription activators and the mechanism whereby contact with these targets stimulates transcription. Using a newly developed method for mapping protein-protein contacts within large complexes, we will identify direct targets of activation domains in several model activators in the presence or absence of chromatin. Biochemical and molecular genetic studies will be conducted to test the relevance of these interactions in transcription. We will extend these studies to examine the targets of a different activator class acting at TATA-less promoters. After mapping these activator-target interactions by hydroxyl radical cleavage and direct protein-protein interaction assays, we will determine the structure of the activation domains in combination with their relevant targets. Finally, by blocking assembly of the Preinitiation Complex at intermediate stages, we will examine the mechanism by which activator contact with 1 of these targets, the SAGA coactivator complex, stimulates transcription. Our proposed work will illuminate important mechanisms and principles of transcriptional regulation.
Keywords: DNA directed RNA polymerase, fungal genetics, genetic promoter element, genetic regulation, genetic transcription, molecular genetics, protein protein interaction, antibody, chemical cleavage, chromatin, crosslink, enzyme structure, hydroxyl radical, molecular site, protein binding, ribosomal protein, transcription factor, SDS polyacrylamide gel electrophoresis, Saccharomyces cerevisiae, animal tissue
Project start date: 2005-09-01
Project end date: 2009-08-31
1R01GM075114-01 (2005): $473082
Molecular Analysis Of Eukaryotic Transcription
Steven M Hahn, Member
Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 981091024
Grant 5R01GM053451-12 from National Institute Of General Medical Sciences IRG: MGB
Project start date: 1995-09-30
Project end date: 2009-03-31
5R01GM053451-12 (2007): $665170
MOLECULAR ANALYSIS OF RNAPII TRANSCRIPTION INITIATION
Steven M Hahn, Member
Fred Hutchinson Cancer Research Center
box 19024, 1100 Fairview Ave N
seattle, Wa 981091024
Grant 5R01GM042551-08 from National Institute Of General Medical Sciences IRG: MBY
Project start date: 1989-07-01
Project end date: 1998-06-30
5R01GM042551-08 (1996): $310302
MOLECULAR ANALYSIS OF RNAPIII TRANSCRIPTION INITIATION
Steven M Hahn, Member
Fred Hutchinson Cancer Research Center
box 19024, 1100 Fairview Ave N
seattle, Wa 981091024
Grant 5R01GM053451-02 from National Institute Of General Medical Sciences IRG: MBY
Project start date: 1995-09-30
Project end date: 2000-03-31
5R01GM053451-02 (1996): $256159