A multidisciplinary study aimed at uncovering the structural and energetic basis for transcriptional activation in the E. coli pap operon is proposed. Genetic, biochemical and X-ray crystallographic experiments will be employed to test an elegant model for regulation known as the phase variation control mechanism. This model invokes the DNA methylation state of two upstream GATC sequences as a key factor determining which of six possible binding sites are occupied by the leucine-responsive regulatory protein (Lrp). The translocation of Lrp dimers along the DNA, in a process mediated by the coactivator protein PapI, is essential for RINA polymerase binding and subsequent transcription. Rigorous biochemical experiments will provide a comprehensive set of binding free-energy parameters describing the inherent DNA sequence preference of the Lrp protein for individual sites, together with how this is modulated by the physiologically relevant effectors. Several in vivo genetic selections will also be applied to Lrp, to identify specific amino acids involved in the responsiveness to DNA methylation and to PapI binding. Finally, X-ray crystallography will be used to determine the high-resolution atomic structure of unliganded Lrp, of the binary Lrp-DNA complexes with several different single-sites, and of the ternary Lrp-PapI-DNA complex. The analyses promise considerable general insight into the basis of methylation effects on protein-DNA interactions, as well as the interplay of protein-induced and intrinsic A-tract bending in the formation of higher-order nucleoprotein structure. The pili proteins regulated by this operon are essential for targeting of bacteria to the surface of uroepithelial cells lining the human urinary tract. Detailed information on pap regulation should thus be helpful in designing strategies to inhibit host colonization and pathogenicity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062630-02
Application #
6621909
Study Section
Biochemistry Study Section (BIO)
Program Officer
Flicker, Paula F
Project Start
2002-01-01
Project End
2005-12-31
Budget Start
2003-01-01
Budget End
2003-12-31
Support Year
2
Fiscal Year
2003
Total Cost
$253,240
Indirect Cost
Name
University of California Santa Barbara
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
094878394
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106