Protein recognition of specific DNA sequences is critical for the proper regulation of transcription. Binding specificity is accomplished through a series of direct and solvent-mediated protein-DNA contacts. The intrinsic sequence-dependent ability of DNA to assume specific conformations can also play a role in specificity. Small molecule effectors, acting as an environmental signals, may bind to the regulator to induce needed conformational changes for specific DNA binding. Recognizing such signals is crucial for precise transcription regulation and requires effector binding to display high affinity. Improper spatial or temporal transcription can lead to uncontrolled cellular proliferation or cell death. Thus, the complete elucidation of the mechanisms by which environmental signals are transduced into the biological response of transcription repression requires the combination of crystallographic, biochemical and thermodynamic studies. As a model system, we are continuing our structural and biochemical studies on the E. coli Purine Repressor (PurR), and allosterically regulated transcription repressor. The five specific aims are to: (1) Extend the resolution of the PurR-hypoxanthine/guanine-purF (and purF (pal) operator ternary complexes from 2.7 cross of the circle to at least 2.2. cross of the circle resolution and the corepressor-free CBD to at lest 2.0 cross of the circle resolution. (2) Determine the structures, binding affinities and thermodynamic properties of PurR-corepressor-purF operator complexes in which PurR residues involved in major groove binding, Thr15, Thr16, His20 and Arg20, or minor groove binding, Val50, Ala51, Ser53, Leu54, Lys55 and Val56, are substituted. (3) Determine the structures and DNA and corepressor binding affinities of PurR-corepressor-purF operator complexes in which PurR residues Arg190, Thr192 and Glu222, which line the corepressor binding pocket, are substituted. (4) Determine the structures and DNA and corepressor binding affinities of PurR-corepressor-ppurF operator complexes and the structures of the unliganded Corepressor Binding Domain (residues 53-341) of PurR in which residues Tyr 73, Phe74, Tyr107, Trp147, Asp160, Asn161 and Gln292 are substituted. (5) Crystallize and determine the structures and binding affinities of PurR- hypoxanthine-DNA operator complexes (a) in which purF operator base pair A8:T8' has been replaced by a T:A, C:G or G:C base pair or (b) in which the purF central C9pG9' base pair step is replaced with CpI, TpDAP, TpAP, dUpDAP, dUpAP, GpC and ApT and TpA steps.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM049244-04
Application #
2405236
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1994-08-01
Project End
2001-07-31
Budget Start
1997-08-01
Budget End
1998-07-31
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
Allen, Gregory S; Steinhauer, Katrin; Hillen, Wolfgang et al. (2003) Crystal structure of HPr kinase/phosphatase from Mycoplasma pneumoniae. J Mol Biol 326:1203-17
Steinhauer, Katrin; Allen, Gregory S; Hillen, Wolfgang et al. (2002) Crystallization, preliminary X-ray analysis and biophysical characterization of HPr kinase/phosphatase of Mycoplasma pneumoniae. Acta Crystallogr D Biol Crystallogr 58:515-8
Huffman, Joy L; Lu, Fu; Zalkin, Howard et al. (2002) Role of residue 147 in the gene regulatory function of the Escherichia coli purine repressor. Biochemistry 41:511-20
Schumacher, Maria A; Pearson, Robert F; Moller, Thorleif et al. (2002) Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein. EMBO J 21:3546-56
Huffman, J L; Mokashi, A; Bachinger, H P et al. (2001) The basic helix-loop-helix domain of the aryl hydrocarbon receptor nuclear transporter (ARNT) can oligomerize and bind E-box DNA specifically. J Biol Chem 276:40537-44
Brown, M; Schumacher, M A; Wiens, G D et al. (2000) The structural basis of repertoire shift in an immune response to phosphocholine. J Exp Med 191:2101-12
Zheleznova, E E; Markham, P N; Neyfakh, A A et al. (1999) Structural basis of multidrug recognition by BmrR, a transcription activator of a multidrug transporter. Cell 96:353-62
Glasfeld, A; Koehler, A N; Schumacher, M A et al. (1999) The role of lysine 55 in determining the specificity of the purine repressor for its operators through minor groove interactions. J Mol Biol 291:347-61
Lu, F; Schumacher, M A; Arvidson, D N et al. (1998) Structure-based redesign of corepressor specificity of the Escherichia coli purine repressor by substitution of residue 190. Biochemistry 37:971-82
Lu, F; Brennan, R G; Zalkin, H (1998) Escherichia coli purine repressor: key residues for the allosteric transition between active and inactive conformations and for interdomain signaling. Biochemistry 37:15680-90

Showing the most recent 10 out of 14 publications