MECHANISMS OF COOPERATIVE PROTEIN-DNA INTERACTIONS

Project: Research project

Project Details

Description

The goal of the proposed biophysical studies is to understand the
physical mechanisms by which cooperative DNA-binding proteins
regulate the transcription of genes. Thermodynamic binding
studies of cI-repressor from phage lambda and gal-repressor form
E. coli. to their respective operators will be conducted using the
quantitative DNase footprint titration method. This method is
unique in that it allows resolution of both the intrinsic and
cooperative Gibbs free energies for proteins which bind to
multiple, specific-sites on DNA. The Gibbs free energies will be
used to test statistical mechanical models on the cooperative
interactions.

Analysis of both the cI- and gal-repressors will be conducted in
order to identify common mechanisms of cooperative interactions
in different regulatory systems. The cI-repressor binds to three
adjacent sites on the DNA. The proposed studies will focus on
determining the mechanism of the cooperative interactions among
the three binding sites and determining if the contacts between
the repressor and DNA affect the cooperative interactions. In
contrast, gal-repressor binds to two specific-sites separated by
many turns of the DNA-helix. Studies are proposed to determine
if gal-repressor binds cooperatively to DNA and if so, to
determine the mechanism. Thermodynamic characterization of
the binding and cooperative interactions among gal-repressor,
catabolite activator protein (CAP), and RNA polymerase as a
function of the allosteric regulatory molecules galactose and
cAMP is proposed in order to develop a comprehensive physical-
chemical description of the regulation of the goal operon.

Studies are also proposed to determined whether DNA-topology
affects cooperative binding of both the gal- and cI-repressors.
Further development of the footprint titration method is proposed
in order to directly measure repressor binding to supercoiled
DNA. Other issues to be considered include determining how the
geometric relationship of the repressor binding affects interaction
between sites, and if local changes in DNA structure play a role in
mediating cooperativity.
StatusFinished
Effective start/end date1/1/901/1/90

Funding

  • National Institute of General Medical Sciences

ASJC

  • Genetics
  • Molecular Biology
  • Biophysics
  • Structural Biology
  • Biochemistry

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