A central problem in molecular biology is the understanding of
transcriptional regulation. This understanding requires, as a
first step, the identification of the transcription factors, the
determination of structures by x-ray crystallography, and some
knowledge of the larger structural features of the entire regulatory
complex as well as information concerning the functional roles
of the various proteins. This first step has been reached for
both prokaryotes and eukaryotes for at least a few of the regulatory
proteins, providing the opportunity to proceed with detailed mechanistic
studies employing the mathematical techniques and instrumentation
of molecular biophysics. Experiments are proposed employing time-resolved
fluorescence measurements of energy transfer to unravel mechanistic
details of transcriptional regulation. These studies are directed
toward uncovering structural changes and the associated thermodynamics
of the intermediates in the formation of binary complexes, such
as that formed from the TATA box of DNA and TBP (TATA binding
protein) and in structures of increasing complexity. In addition,
molecular dynamics and Langevin dynamics will be used to fit and
model the time-resolved anisotropy decays of fluorescent probes
tethered to oligonucleotides. The complex chemical kinetics will
be fit by programs developed in-house. These general minimization
programs obtain optimum rate constants and activation energies
using a subroutine that integrates the differential equations
appropriate for a given mechanism. The fitting is global, in
which all data sets (with different initial concentrations and
temperatures) are fit simultaneously. In this manner a kinetic
and thermodynamic profile will be obtained for the initial steps
of transcription. For many of the experiments, measurements to
detect the bending and unwinding of DNA during the transcription-related
reactions will also require prior measurement s of DNA duplex
formation and structural alterations in dsDNA induced by backbone
modifications and sequence changes. |
