The growth, development, and metabolism of healthy cells depends on the precise regulation of gene expression and error-free maintenance of the genome. A detailed understanding of the underlying principles of protein-DNA recognition and protein-DNA architectures is therefore crucial for understanding how diseases can result from failures in these systems. The proposed research addresses the question of how an oligomeric DNA-binding protein, which can in principle adopt a highly symmetric conformation, binds to a DNA recognition element that can be at most twofold symmetric. The tetrameric tumor suppresser p53, the trimeric heat shock transcription factor HSF, and the tetrameric lactose repressor are well-studied examples of such oligomeric proteins. In each case, the protein uses multiple DNA-binding domains to bind sequences composed of direct and inverted repeats of a recognition element. In the proposed research, the hexameric arginine repressor (ArgR) will serve as a model system for studying the architecture of an oligomeric protein-DNA assembly. ArgR plays a multifuctional role in the bacterial cell, serving as the master regulator of the arginine biosynthetic genes as well as playing an obligatory architectural role in mediating site-specific recombination events. In the presence of the corepressor L-arginine, ArgR uses four of its six DNA-binding domains to recognize DNA operators composed of a tandem repeat of palindromic """"""""Arg boxes"""""""". The ArgR DNA-binding domains are members of the winged helix-turn-helix (wHTH) family of DNA-binding motifs. This growing family includes domains found in histone H5, the ets family, HNF- 3/forkhead, and the heat shock transcription factor. In order to build a structural framework for understanding the basic principles of how oligomeric proteins recognize complex DNA sequences, and how multiple wHTH domains interact at the DNA surface, X-ray diffraction methods will be used to determine structures of (i) ArgR in the low affinity DNA-binding state, (ii) ArgR in the high affinity state with bound corepressor, (iii) an ArgR superrepressor, (iv) an ArgR-DNA complex representative of the architectural role of ArgR in site-specific recombination, and (v) an ArgR-DNA complex formed with a tandem Arg box operator.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056980-03
Application #
6342990
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
1999-01-01
Project End
2002-12-31
Budget Start
2001-01-01
Budget End
2001-12-31
Support Year
3
Fiscal Year
2001
Total Cost
$142,218
Indirect Cost
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Wong, Ming-Ching; Dobi, Krista C; Baylies, Mary K (2014) Discrete levels of Twist activity are required to direct distinct cell functions during gastrulation and somatic myogenesis. PLoS One 9:e99553
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