DNA SEQUENCE RECOGNITION BY CAP

Project Details

Description

The catabolite gene activator protein (CAP) of E. coli is a
member of a class of sequence-specific DNA binding proteins that
utilize a highly conserved alpha-helix-turn-alpha-helix
structural motif to mediate interaction with DNA. This class of
sequence-specifc DNA binding proteins has at least 75 members,
including the homeo box proteins, which are involved in the
regulation of eukaryotic development. Our research project
regarding DNA-sequence recognition by CAP is directed at three
long-range objectives: (i) identification of the individual
amino acid-DNA contacts involved in DNA-sequence recognition by
CAP, (ii) investigation of the chemistry and thermodynamics of
specificity, and (iii) elucidation of rules--a "coded"--relating
the amino acid sequence of the helix-turn-helix motif of a DNA
binding protein to the nucleotide sequence of the specific DNA
site. The experiments in this proposal have as specific aims the
investigation of two examples of DNA-sequence recognition
mediated through protein-base pair contacts, and the
investigation of two examples of DNA-sequence recognition
mediated through protein-phosphate contacts. DNA-sequence
recognition mediated through protein-specific DNA binding
protein. The approach to be employed emphasizes the use of single amino
acid substitutions to identify and quantify important amino acid-
DNA interactions. Single amino acid substitutions will be
introduced into CAP using site-directed mutagenesis. The DNA-
sequence recognition properties of the substituted CAP variants
then will be investigated in in vitro binding assays using as
ligands the consensus DNA site and a battery of non-consensus and
chemically modified DNA sites; free energies of binding and free
energies of specificity will be determine from the equilibrium
binding data. The experimentally measured effects of the amino
acid substitutions on the binding and specificity free energies
will be interpreted in two fashions: (i) qualitatively, using
computer-assisted molecule graphics and the known three-
dimensional structures of CAP and B-DNA; and (ii) quantitatively,
using molecular energy calculations. The results to be obtained will be relevant to understanding
protein-nucleic acid interaction and its role in the regulation
of gene expression. In addition, the results to be obtained will
be relevant to understanding other examples of protein-ligand
interaction, including: enzyme-substrate, receptor-hormone, and
receptor-drug interactions.
StatusActive
Effective start/end date12/1/881/31/23

Funding

  • National Institutes of Health: $374,706.00
  • National Institutes of Health: $263,689.00
  • National Institutes of Health: $156,899.00
  • National Institutes of Health: $272,215.00
  • National Institutes of Health: $378,588.00
  • National Institutes of Health: $274,225.00
  • National Institutes of Health: $515,171.00
  • National Institutes of Health: $302,118.00
  • National Institutes of Health: $449,399.00
  • National Institutes of Health: $449,399.00
  • National Institutes of Health
  • National Institutes of Health: $263,689.00
  • National Institutes of Health
  • National Institutes of Health: $515,171.00
  • National Institutes of Health: $452,255.00
  • National Institutes of Health: $374,706.00
  • National Institutes of Health: $263,689.00
  • National Institutes of Health: $269,905.00
  • National Institutes of Health: $41,337.00
  • National Institutes of Health: $303,866.00
  • National Institutes of Health: $371,380.00
  • National Institutes of Health: $266,409.00
  • National Institutes of Health: $48,358.00
  • National Institutes of Health: $450,943.00
  • National Institutes of Health: $302,720.00
  • National Institutes of Health
  • National Institutes of Health: $452,255.00
  • National Institutes of Health: $301,674.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $263,689.00
  • National Institutes of Health: $301,621.00

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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