TIME RESOLVED SYNCHROTRON XRAY "FOOTPRINTING"

  • Brenowitz, Michael D. (PI)
  • Chance, Britton (PI)
  • Miller, Lisa (PI)
  • Blasie, J. Kent (PI)
  • Maleknia, Simin (PI)
  • Goldsmith, Sharon (PI)
  • Pyle, Anna Marie (PI)
  • Toomey, John R. (PI)
  • Franzen, Stephen (PI)
  • Downard, Kevin (PI)
  • Marinkovic, Nebojsa (PI)
  • Sperber, Ellen (PI)
  • Crothers, Donald (PI)
  • Penner-Hahn, James E. (PI)
  • Kravtsov, Irina (PI)
  • Davies, David (PI)
  • Silva, Alberto (PI)
  • Morris, Ann-Tabor (PI)
  • Hurley, J.I.M. (PI)
  • Fisher, Timothy (PI)
  • Seeman, Nadrian C. (PI)
  • Burke, John (PI)
  • Peter, Sun (PI)
  • Wlodawer, Alex (PI)
  • Studier, Bill (PI)
  • Xinhua, Ji (PI)
  • Tran, Alex (PI)
  • Dauter, Zbigniew (PI)
  • Honggao, Yan (PI)
  • Kovacs, Kevin (PI)
  • vanamee, Eva (PI)
  • Snyder, Gary (PI)
  • Que Lawrence, [No Value] (PI)
  • Chen, Terry (PI)
  • Pedersen, Lars (PI)
  • Hurley, James (PI)
  • Sonenberg, Nils (PI)
  • Anderson, Richard (PI)
  • Bubacco, Luigi (PI)
  • Sharpe, L.E.E. (PI)
  • Kallenbach, Neville (PI)
  • Aihua, Xie (PI)
  • Caffrey, Martin (PI)
  • Sinclair, Robert (PI)
  • Woodson, Sarah (PI)
  • Kwok, Yue (PI)
  • Blasie, J. Kent (PI)
  • Lawrence, Que (PI)
  • Heiney, Paul (PI)
  • Anni, Helen (PI)
  • Holmes, Kenneth (PI)
  • Powers, Linda (PI)
  • Scheuring, E.V.A. (PI)
  • Scarrow, Robert (PI)
  • Chance, Mark (PI)
  • Sagi, Irit (PI)
  • Maroney, Michael (PI)
  • Sullivan, Michael (PI)
  • Caroline Caroline, Lee (PI)
  • Penner-Hahn, James E. (PI)
  • Blasie, J. Kent (PI)
  • Hurley, J.I.M. (PI)
  • Seeman, Nadrian C. (PI)
  • Toomey, John R. (PI)
  • Que Lawrence, [No Value] (PI)
  • Bubacco, Luigi (PI)
  • Sharpe, L.E.E. (PI)
  • Kallenbach, Neville (PI)
  • Aihua, Xie (PI)
  • Caffrey, Martin (PI)
  • Sinclair, Robert (PI)
  • Woodson, Sarah (PI)
  • Kwok, Yue (PI)
  • Blasie, J. Kent (PI)
  • Lawrence, Que (PI)
  • Heiney, Paul (PI)
  • Anni, Helen (PI)
  • Holmes, Kenneth (PI)
  • Powers, Linda (PI)
  • Scheuring, E.V.A. (PI)
  • Scarrow, Robert (PI)
  • Chance, Mark (PI)
  • Sagi, Irit (PI)
  • Maroney, Michael (PI)
  • Sullivan, Michael (PI)
  • Caroline Caroline, Lee (PI)
  • Almo, Steven C. (PI)

Project: Research project

Project Details

Description

Hydroxyl radicals (OH) can cleave the phosphodiester backbone of
nucleic acids and are valuable reagents in the study of nucleic acid
structure and protein-nucleic acid interactions. Irradiation of
solutions by high flux "white light" x-ray beams based on bending
magnet beamlines at the National Synchrotron Light Source (NSLS)
yields sufficient concentrations of OH so that quantitative nuclease
protection ("footprinting") studies of DNA and RNA can be conducted
with a duration of exposure in the range of 50 - 100 msec. The
sensitivity of DNA and RNA to x-ray mediated OH cleavage is
equivalent. Both nucleic acids are completely protected from
synchrotron x-ray induced cleavage by the presence of thiourea in the
sample solution, demonstrating that cleavage is mediated exclusively
by free radicals. The utility of this time-dependent approach to
"footprinting" is demonstrated by obtaining a synchrotron x-ray
"footprint" of a protein-DNA complex and by a time-resolved
"footprinting" analyses of the Mg2+-dependent folding of the
Tetrahymena thermophilia L-21 Sca I ribozyme RNA. Equilibrium
titrations reveal differences among the ribozyme domains in the
cooperativity of Mg2+-dependent folding. RNA folding progress curves
over timescales of 30 sec. to several minutes were obtained for
several regions of the ribozyme. The rate constants obtained for the
P3-P7, P4-P6 and P5 domains are comparable with those obtained by
Zarrinkar & Williamson [Science 265: 918-924, 1994] using a
hybridization-competition assay and confirm the hierarchy of the
folding of the ribozyme domains established by these investigators.
The folding rates obtained for individual regions within domains
suggest additional steps in the folding pathway. Synchrotron x-ray
"footprinting" is a new approach of general applicability for the
study of time-resolved structural changes of nucleic acid conformation
and protein-nucleic acid complexes.
StatusFinished
Effective start/end date1/1/018/31/03

Funding

  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources
  • National Center for Research Resources

ASJC

  • Radiation
  • Genetics
  • Molecular Biology
  • Structural Biology
  • Biophysics
  • Spectroscopy
  • Biochemistry
  • Microbiology
  • Ecology, Evolution, Behavior and Systematics
  • Computer Science(all)
  • Software
  • Clinical Neurology
  • Nephrology
  • Neurology
  • Information Systems
  • Infectious Diseases
  • Polymers and Plastics
  • Medicine(all)
  • Geometry and Topology
  • Electrical and Electronic Engineering
  • Rheumatology
  • Immunology
  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Fluid Flow and Transfer Processes
  • Oncology
  • Cancer Research
  • Biotechnology
  • Hardware and Architecture
  • Catalysis
  • Cell Biology
  • Statistics, Probability and Uncertainty
  • Pharmaceutical Science

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