Transcriptional gene regulation is an intense field of research in biology and in medicine. The lac operon of E. coli is the paradigm for transcriptional gene regulation. Its key component, the lac repressor (lacR), is one of the most intensely studied proteins both genetically and biochemically. Recent crystal structures of the uncomplexed tetrameric lacR, lacR bound to the inducer IPTG, and lacR bound to operator DNA have provided a framework for understanding how lacR functions as a genetic switch. The research in this proposal will extend our current structural understanding of lacR. Of paramount importance is extending the resolution of the structure of lacR bound to operator DNA. Since the structure was determined at only 4.8Angstrom units resolution, the current model for the operator-bound, repressed conformation of lacR does not include side chains. A major premise of this proposal is that a dimeric version of lacR, when complexed to operator DNA, will grow crystals which diffract to higher resolution than has so far been achieved for the tetramer of lacR. Dimeric lacR will also be used to achieve: (1) a structural comparison of lacR bound to both wild-type and symmetric operator sequences, (2) a structural basis for the action of allolactose, the natural inducer of lacR, and orthonitrophenylfucoside, an anti-inducer of lacR, and (3) a structural basis for single point mutations in lacR which lead to 100-fold increased affinity for operator.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM019250-03
Application #
6150990
Study Section
Special Emphasis Panel (ZRG3-BBCA (03))
Program Officer
Cassatt, James
Project Start
2000-01-06
Project End
Budget Start
2000-01-06
Budget End
2001-01-05
Support Year
3
Fiscal Year
2000
Total Cost
$39,232
Indirect Cost
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104