Difference: SethDarst (1 vs. 8)

• Name: Seth Darst
• Email: darst@rockefeller.edu
• Company Name: Rockefeller University
• URL: http://www.rockefeller.edu/research/abstract.php?id=32
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Grant Number: 5R01GM081697-02 Project Title: Structural Basis for Sporulation in Bacillus PI Information: Name Email Title DARST, SETH A. darst@rockefeller.edu PROFESSOR

Abstract: DESCRIPTION (provided by applicant): Sporulation in Bacillus subtilis has been studied extensively as a model for cellular development. Initiation of sporulation depends on sensor histidine-kinases (His-kinases), which autophosphorylate, initiating phosphate transfer through an expanded two-component signaling system, the phosphorelay. Subsequent stages of sporulation are controlled by a cascade of sporulation-specific RNA polymerase ??factors, which is initiated by ?F. The activity of ?F is regulated in a compartment-specific manner by a complex network of accessory factors, including the SpollE^? phosphatase. Here, we propose biochemical and structural approaches to address the structure/function relationship for two key regulators of Bacillus sporulation, the sensor His-kinase KinB, and SpollE^?, using the thermophile B. stearothermophilus (Bst) as a model system for B. subtilis and the category A pathogen B. anthracis. Specifically, we propose to: 1. Determine the crystal structure of the Bst Sda/KinB complex. Sda is a 46-residue polypeptide that inhibits KinB in response to DNA damage. We have obtained crystals of the soluble, cytoplasmic catalytic core of KinB bound to Sda that diffract beyond 2 A-resolution. 2. Determine the crystal structure of KinB trapped in the act of autophosphorylation. The catalytic core of the His-kinases comprises two domains, the His-phosphotransferase domain (Hpt; harboring the phospho-acceptor His residue) and the ATP-binding kinase domain. A large scale conformational change is required to reposition the kinase domain with respect to the Hpt domain for autophosphorylation to occur. We propose two strategies to trap this transient conformational state for structural studies.; 3. Determine crystal structures of Sda/KinB/Spo0F complexes. To understand the structural basis for phosphotransfer from the Hpt to the response regulator Spo0F, we propose to solve the ternary complex structure with unphosphorylated KinB (preliminary crystals have been obtained), as well as with a stable phospho-His analog, (4'-phospho-2'-furyl)alanine, which will be incorporated into the KinB Hpt by expressed protein ligation.; 4. Structurally characterize the ?F regulatory system (?F/SpollAA/SpollAB/SpollE) controlling the initiation of Bacillus sporulation.

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Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100656399 Fiscal Year: 2008 Department: MOLECULAR BIOPHYSICS LAB Project Start: 28-SEP-2007 Project End: 31-JUL-2011 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: PCMB

Abstract: DESCRIPTION (provided by applicant): TRCF (Transcription Repair Coupling Factor) is a widely conserved bacterial protein that couples DNA repair with transcription. TRCF recognizes RNAP stalled at a non-coding template site of DNA damage, disrupts the transcription complex to release the transcript and RNAP, and recruits the DNA excision repair machinery to the site. The mechanism of RNA release has been illuminated by the discovery that TRCF causes forward translocation of RNAP, using an ATP-dependent motor that is highly homologous to that of the Holliday branch migration protein RecG. TRCF is a large (130 kDa), multi-functional protein with a complex structure/function relationship that is currently understood only from sequence analysis and genetic manipulation. In this grant, we propose detailed structural studies to elucidate the structure/function relationship of TRCF, to reveal conformational changes involved in the ATP-hydrolysis cycle and its coupling to the DNA translocase activity, and to reveal the interactions of TRCF with the RNAP ternary elongation complex. Specifically, we propose to: 1. Determine the X-ray crystal structure of TRCF. 2. Determine the structural basis for TRCF DNA translocase activity through X-ray crystal structures with nucleotides and/or nucleotide analogs. 3. Determine the structural basis for the specific transcription termination activity of TRCF through structural studies of an RNAP ternary elongation complex (RNAP/DNA/RNA) with TRCF.

Thesaurus Terms:

Abstract: DESCRIPTION (provided by applicant): In bacteria, the 450 kDa RNA polymerase (RNAP) holoenzyme, comprising the evolutionarily conserved catalytic core (subunit composition alpha2betabeta'somega) combined with the initiation-specific sigma subunit, directs transcription initiation. Bacterial transcription depends on a primary sigma factor that is essential for viability, as well as alternative sigma's that control specific regulons. A major mechanism to control transcription initiation is through regulation of sigma activity. Dramatic insights have come from structural studies of sigma's and holoenzymes. Nevertheless many challenges remain. In this competing continuation, we propose studies to further our understanding of sigma factor structure and function, and interactions of accessory factors. Specifically, we propose to: 1. Characterize sigma factor structure and function. We will: a) Determine the structural basis for sigma interactions with the -10 element, b) Determine the structural basis for -35 element recognition by an alternative sigma, c) Probe the solution conformation of free ? using disulfide crosslinking, and d) Probe interdomain interactions of free sigma using segmental labeling and solution NMR. 2. Structurally characterize sigma/anti-sigma complexes. We will determine structures of: a) R. sphaeroides sigma/E/ChrR, and b) E. coli sigma/32/DnaK. 3. Structurally characterize interactions involved in transcription activation. We will: a) Investigate the bacteriophage lambda cl protein and the mechanism of activation, b) Investigate the role of the bacteriophage lambda cll protein in activation, and c) Determine the structure of the B. subtilis Spx/alpha-C-terminal-domain complex. 4. Structurally characterize the sigmaF regulatory system (sigmaF/SpollAA/SpollAB/SpollE) controlling the initiation of sporulation in Bacillus.

Thesaurus Terms: DNA directed RNA polymerase, bacterial protein, intermolecular interaction, protein structure function, transcription factor bacterial virus, enzyme structure, genetic promoter element Bacillus subtilis, Escherichia coli, Rhodospirillales, X ray crystallography, nuclear magnetic resonance spectroscopy

• Name: Seth Darst
• Email: darst@rockefeller.edu
• Company Name: Rockefeller University
• URL: http://www.rockefeller.edu/research/abstract.php?id=32
• Location: RockefellerUOffice
• Country: USA
• Comment:

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Grant Number: 1R01GM073829-01 PI Name: DARST, SETH A. PI Email: darst@rockefeller.edu PI Title: PROFESSOR Project Title: Structure/Function of the Bacterial TRCF

Abstract: DESCRIPTION (provided by applicant): TRCF (Transcription Repair Coupling Factor) is a widely conserved bacterial protein that couples DNA repair with transcription. TRCF recognizes RNAP stalled at a non-coding template site of DNA damage, disrupts the transcription complex to release the transcript and RNAP, and recruits the DNA excision repair machinery to the site. The mechanism of RNA release has been illuminated by the discovery that TRCF causes forward translocation of RNAP, using an ATP-dependent motor that is highly homologous to that of the Holliday branch migration protein RecG. TRCF is a large (130 kDa), multi-functional protein with a complex structure/function relationship that is currently understood only from sequence analysis and genetic manipulation. In this grant, we propose detailed structural studies to elucidate the structure/function relationship of TRCF, to reveal conformational changes involved in the ATP-hydrolysis cycle and its coupling to the DNA translocase activity, and to reveal the interactions of TRCF with the RNAP ternary elongation complex. Specifically, we propose to: 1. Determine the X-ray crystal structure of TRCF. 2. Determine the structural basis for TRCF DNA translocase activity through X-ray crystal structures with nucleotides and/or nucleotide analogs. 3. Determine the structural basis for the specific transcription termination activity of TRCF through structural studies of an RNAP ternary elongation complex (RNAP/DNA/RNA) with TRCF.

Thesaurus Terms:

Abstract: DESCRIPTION (provided by applicant): In bacteria, the 450 kDa RNA polymerase (RNAP) holoenzyme, comprising the evolutionarily conserved catalytic core (subunit composition alpha2betabeta'somega) combined with the initiation-specific sigma subunit, directs transcription initiation. Bacterial transcription depends on a primary sigma factor that is essential for viability, as well as alternative sigma's that control specific regulons. A major mechanism to control transcription initiation is through regulation of sigma activity. Dramatic insights have come from structural studies of sigma's and holoenzymes. Nevertheless many challenges remain. In this competing continuation, we propose studies to further our understanding of sigma factor structure and function, and interactions of accessory factors. Specifically, we propose to: 1. Characterize sigma factor structure and function. We will: a) Determine the structural basis for sigma interactions with the -10 element, b) Determine the structural basis for -35 element recognition by an alternative sigma, c) Probe the solution conformation of free ? using disulfide crosslinking, and d) Probe interdomain interactions of free sigma using segmental labeling and solution NMR. 2. Structurally characterize sigma/anti-sigma complexes. We will determine structures of: a) R. sphaeroides sigma/E/ChrR, and b) E. coli sigma/32/DnaK. 3. Structurally characterize interactions involved in transcription activation. We will: a) Investigate the bacteriophage lambda cl protein and the mechanism of activation, b) Investigate the role of the bacteriophage lambda cll protein in activation, and c) Determine the structure of the B. subtilis Spx/alpha-C-terminal-domain complex. 4. Structurally characterize the sigmaF regulatory system (sigmaF/SpollAA/SpollAB/SpollE) controlling the initiation of sporulation in Bacillus.

Thesaurus Terms: DNA directed RNA polymerase, bacterial protein, intermolecular interaction, protein structure function, transcription factor bacterial virus, enzyme structure, genetic promoter element Bacillus subtilis, Escherichia coli, Rhodospirillales, X ray crystallography, nuclear magnetic resonance spectroscopy

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2005 Department: LAB/MOLECULAR BIOPHYSICS Project Start: 01-MAR-1996 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: PCMB

Grant Number: 1R01GM073829-01 PI Name: DARST, SETH A. PI Email: darst@rockefeller.edu PI Title: PROFESSOR Project Title: Structure/Function of the Bacterial TRCF

Abstract: DESCRIPTION (provided by applicant): TRCF (Transcription Repair Coupling Factor) is a widely conserved bacterial protein that couples DNA repair with transcription. TRCF recognizes RNAP stalled at a non-coding template site of DNA damage, disrupts the transcription complex to release the transcript and RNAP, and recruits the DNA excision repair machinery to the site. The mechanism of RNA release has been illuminated by the discovery that TRCF causes forward translocation of RNAP, using an ATP-dependent motor that is highly homologous to that of the Holliday branch migration protein RecG. TRCF is a large (130 kDa), multi-functional protein with a complex structure/function relationship that is currently understood only from sequence analysis and genetic manipulation. In this grant, we propose detailed structural studies to elucidate the structure/function relationship of TRCF, to reveal conformational changes involved in the ATP-hydrolysis cycle and its coupling to the DNA translocase activity, and to reveal the interactions of TRCF with the RNAP ternary elongation complex. Specifically, we propose to: 1. Determine the X-ray crystal structure of TRCF. 2. Determine the structural basis for TRCF DNA translocase activity through X-ray crystal structures with nucleotides and/or nucleotide analogs. 3. Determine the structural basis for the specific transcription termination activity of TRCF through structural studies of an RNAP ternary elongation complex (RNAP/DNA/RNA) with TRCF.

Thesaurus Terms:

Abstract: DESCRIPTION (provided by applicant): In bacteria, the 450 kDa RNA polymerase (RNAP) holoenzyme, comprising the evolutionarily conserved catalytic core (subunit composition alpha2betabeta'somega) combined with the initiation-specific sigma subunit, directs transcription initiation. Bacterial transcription depends on a primary sigma factor that is essential for viability, as well as alternative sigma's that control specific regulons. A major mechanism to control transcription initiation is through regulation of sigma activity. Dramatic insights have come from structural studies of sigma's and holoenzymes. Nevertheless many challenges remain. In this competing continuation, we propose studies to further our understanding of sigma factor structure and function, and interactions of accessory factors. Specifically, we propose to: 1. Characterize sigma factor structure and function. We will: a) Determine the structural basis for sigma interactions with the -10 element, b) Determine the structural basis for -35 element recognition by an alternative sigma, c) Probe the solution conformation of free ? using disulfide crosslinking, and d) Probe interdomain interactions of free sigma using segmental labeling and solution NMR. 2. Structurally characterize sigma/anti-sigma complexes. We will determine structures of: a) R. sphaeroides sigma/E/ChrR, and b) E. coli sigma/32/DnaK. 3. Structurally characterize interactions involved in transcription activation. We will: a) Investigate the bacteriophage lambda cl protein and the mechanism of activation, b) Investigate the role of the bacteriophage lambda cll protein in activation, and c) Determine the structure of the B. subtilis Spx/alpha-C-terminal-domain complex. 4. Structurally characterize the sigmaF regulatory system (sigmaF/SpollAA/SpollAB/SpollE) controlling the initiation of sporulation in Bacillus.

Thesaurus Terms: DNA directed RNA polymerase, bacterial protein, intermolecular interaction, protein structure function, transcription factor bacterial virus, enzyme structure, genetic promoter element Bacillus subtilis, Escherichia coli, Rhodospirillales, X ray crystallography, nuclear magnetic resonance spectroscopy

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2005 Department: LAB/MOLECULAR BIOPHYSICS Project Start: 01-MAR-1996 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: PCMB

• Name: Seth Darst
• Email: darst@rockefeller.edu
• Company Name: Rockefeller University
• URL: http://www.rockefeller.edu/research/abstract.php?id=32
• Location: RockefellerUOffice
• Country: USA
• Comment:

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Grant Number: 1R01GM073829-01 PI Name: DARST, SETH A. PI Email: darst@rockefeller.edu PI Title: PROFESSOR Project Title: Structure/Function of the Bacterial TRCF

Abstract: DESCRIPTION (provided by applicant): TRCF (Transcription Repair Coupling Factor) is a widely conserved bacterial protein that couples DNA repair with transcription. TRCF recognizes RNAP stalled at a non-coding template site of DNA damage, disrupts the transcription complex to release the transcript and RNAP, and recruits the DNA excision repair machinery to the site. The mechanism of RNA release has been illuminated by the discovery that TRCF causes forward translocation of RNAP, using an ATP-dependent motor that is highly homologous to that of the Holliday branch migration protein RecG. TRCF is a large (130 kDa), multi-functional protein with a complex structure/function relationship that is currently understood only from sequence analysis and genetic manipulation. In this grant, we propose detailed structural studies to elucidate the structure/function relationship of TRCF, to reveal conformational changes involved in the ATP-hydrolysis cycle and its coupling to the DNA translocase activity, and to reveal the interactions of TRCF with the RNAP ternary elongation complex. Specifically, we propose to: 1. Determine the X-ray crystal structure of TRCF. 2. Determine the structural basis for TRCF DNA translocase activity through X-ray crystal structures with nucleotides and/or nucleotide analogs. 3. Determine the structural basis for the specific transcription termination activity of TRCF through structural studies of an RNAP ternary elongation complex (RNAP/DNA/RNA) with TRCF.

Thesaurus Terms:

There are no thesaurus terms on file for this project.

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2005 Department: LAB OF MOLECULAR BIOPHYSICS Project Start: 01-MAY-2005 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: BBCA

Abstract: DESCRIPTION (provided by applicant): In bacteria, the 450 kDa RNA polymerase (RNAP) holoenzyme, comprising the evolutionarily conserved catalytic core (subunit composition alpha2betabeta'somega) combined with the initiation-specific sigma subunit, directs transcription initiation. Bacterial transcription depends on a primary sigma factor that is essential for viability, as well as alternative sigma's that control specific regulons. A major mechanism to control transcription initiation is through regulation of sigma activity. Dramatic insights have come from structural studies of sigma's and holoenzymes. Nevertheless many challenges remain. In this competing continuation, we propose studies to further our understanding of sigma factor structure and function, and interactions of accessory factors. Specifically, we propose to: 1. Characterize sigma factor structure and function. We will: a) Determine the structural basis for sigma interactions with the -10 element, b) Determine the structural basis for -35 element recognition by an alternative sigma, c) Probe the solution conformation of free ? using disulfide crosslinking, and d) Probe interdomain interactions of free sigma using segmental labeling and solution NMR. 2. Structurally characterize sigma/anti-sigma complexes. We will determine structures of: a) R. sphaeroides sigma/E/ChrR, and b) E. coli sigma/32/DnaK. 3. Structurally characterize interactions involved in transcription activation. We will: a) Investigate the bacteriophage lambda cl protein and the mechanism of activation, b) Investigate the role of the bacteriophage lambda cll protein in activation, and c) Determine the structure of the B. subtilis Spx/alpha-C-terminal-domain complex. 4. Structurally characterize the sigmaF regulatory system (sigmaF/SpollAA/SpollAB/SpollE) controlling the initiation of sporulation in Bacillus.

Thesaurus Terms: DNA directed RNA polymerase, bacterial protein, intermolecular interaction, protein structure function, transcription factor bacterial virus, enzyme structure, genetic promoter element Bacillus subtilis, Escherichia coli, Rhodospirillales, X ray crystallography, nuclear magnetic resonance spectroscopy

Institution: ROCKEFELLER UNIVERSITY NEW YORK, NY 100216399 Fiscal Year: 2005 Department: LAB/MOLECULAR BIOPHYSICS Project Start: 01-MAR-1996 Project End: 30-APR-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: PCMB

• Name: Seth Darst
• Email: darst@rockefeller.edu
• Company Name: Rockefeller University
• URL: http://www.rockefeller.edu/research/abstract.php?id=32
• Location: RockefellerUOffice
• Country: USA
• Comment:

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• Name: Seth Darst
• Email: darst@rockefeller.edu
• Company Name: Rockefeller University

• Location: RockefellerUOffice
• Country: USA
• Comment:

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• Name: Seth Darst
• Email: darst@rockefeller.edu
• Company Name: Rockefeller University
• Company URL: http://www.rockefeller.edu
• Location: RockefellerUOffice
• Country: USA
• Comment:

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