Contributing investigators: Mark R. Chance, Gauri Dhavan, Qin He, Sergei
Khrapunov,
Stephanie Morris, Michael Sullivan, Keiji Takamoto and Takeshi Uchida
at the Albert
Einstein College of Medicine and Kay Parkhurst, Lawrence Parkhurst,
Robyn Powell
and Jiong Wu at the University of Nebraska
Abstract
The methods used to study the structure of, and ligand binding by,
macromole-
cules such as proteins and nucleic acids can be broadly grouped into
those that report
global or local (i.e. site-specific) properties. Hydroxyl radical 'footprinting'
allows the
solvent accessibility of the individual sugars of DNA and RNA and individual
residues of
proteins to be quantitated. Equilibrium studies using hydroxyl radical
yield individual-
site isotherms while time-resolved studies conducted using X-ray radiolysis
to produce
the hydroxyl radicals yields individual-site progress curves. Analytical
ultracentrifuga-
tion and small angle X-ray scattering provide no site-specific information,
but rather
report upon the global conformation of a macromolecule. Analysis of
the Mg2+ de-
pendent folding of the Tetrahymena thermophila ribozyme RNA will be
used illustrate
the separate and concordant application of these global and local probes
of macromo-
lecular structure and interaction.
A multifaceted approach is also being used to study the association
of the TATA
Binding Protein (TBP) from Saccharomyces cerevisiae with specific sequences
of DNA.
In this case, hydroxyl radical footprinting together with molecular
dynamics simula-
tions is used to locally probe the TBP-DNA interaction. Changes in
the fluorescence
resonance energy transfer (FRET) of end-labeled DNA upon TBP binding
are used to
globally monitor the extent of protein-induced DNA bending. Intrinsic
protein fluores-
cence and a novel radiolytic protein footprinting assay are being used
to monitor con-
formational changes of local regions of the protein upon DNA binding.
An attempt will
be made to meld these separate views of the TBP-DNA complex into a
portrait that
provides insight into the biology of transcription.
Selected References from our laboratory:
'Pdf' files of these papers can be downloaded from our web page:
<http://www.bioc.aecom.yu.edu/labs/brenlab/Brenowitz-Michael.html>
Parkhurst, K., Brenowitz, M. & Parkhurst, L.J. (1996) Simultaneous
binding and bending of
Promoter DNA by TBP: Real Time Kinetic Measurements. Biochemistry 35,
7459 - 7465.
Petri, V., Hsieh, M., Jamison, E. & Brenowitz, M. (1998) DNA Sequence-specific
recognition
by the 'TATA' Binding Protein: Promoter dependent differences in the
thermodynamics and
kinetics, Biochemistry 37, 15842 - 15849.
Sclavi, B., Chance, M., Brenowitz, M. & Woodson, S. (1998) Visualizing
RNA Folding at Milli-
second Intervals by Synchrotron Hydroxyl Radical Footprinting. Science
279, 1940 - 1943.
Pastor, N., Weinstein, H., Jamison, E. & Brenowitz, M. (2000) A
Detailed Interpretation of
OH Radical Footprints in a TBP-DNA Complex Reveals the Role of Dynamics
in the Mecha-
nism of Sequence-Specific Binding, J. Mol. Biol., 304, 55 - 68.
Wu, J., Parkhurst, K.M., Powell, R.M., Brenowitz, M. & Parkhurst,
L.J. (2001) DNA bends in
solution TBP-TATA complexes are sequence dependent and highly correlated
with tran-
scription activity, J. Biol. Chem. 276, 14614 - 14622.
Dhavan, G.M., Crothers, D.M., Chance, M.R. and Brenowitz, M. (2002)
Concerted Binding
and Bending during IHF-DNA Complex Formation by Time-resolved X-ray
Hydroxyl Radical
Footprinting, J. Mol. Biol. 315, 1027 - 1037.