Difference: SethDarst (5 vs. 6)

• 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