The objective of this project is to elucidate the molecular mechanisms by which the expression of genetic information is controlled in biology. One aspect of the work focuses on the recent discovery that messenger RNAs from genes that encode metabolic enzymes contain binding sites for small molecule metabolites which are related to the function of the enzyme. Binding of the metabolite can turn gene expression on or off at the transcriptional or translational level. Our work on this project focuses on understanding how kinetic and equilibrium factors determine the properties of the"""""""" riboswitch"""""""", using steady state and stopped flow kinetic techniques with fluorescence detecton. Riboswitches that respond to flavin mononucleotide (FMN), adenine, and glycine are the current targets of study. A second aspect of the work focuses on identifying and characterizing the rate of the DNA-bending reaction step in the formation of protein-DNA complexes. Proteins studied include E. coli integration host factor (IMF), which acts to produce a U-turn bend in DNA at specific sites of recombination, and E. coli MutS protein, which recognizes and bends DNA at damage sites in the first step of the process of DNA repair. The bending reaction is followed by fluorescence resonance energy transfer (FRET) between donor and acceptor molecules attached to DNA on opposite sides of the bend site. A third aspect of the work focuses on understanding the equilibria and mechanism of binding p53 tumor suppressor protein to DNA, with particular emphasis on the contribution of non-contacted spacer DNA sequences between binding sites. p53, mutations which are present in nearly half of human tumors, binds DNA as a tetramer, but sites with variable distance between the two dimer binding sites appear to function as transcriptional regulators. Our work includes measuring binding equilibria and kinetics for reaction with a series of DNA sites, starting with the p53 DNA binding domain, and continuing to larger molecules that include the tetramer interface, and ultimately the intact protein. We use the method of DNA cyclization kinetics to characterize the curvature and flexibility of the individual sequences, with and without protein bound. Using a statistical mechanical theory, we relate the free DNA properties to the variation in equilibrium binding constant for the different sites. The long term potential medical impact of this work is to provide for rational intervention in control of gene expression to combat disease.
| Xi, Zhiqun; Zhang, Yongli; Hegde, Rashmi S et al. (2010) Anomalous DNA binding by E2 regulatory protein driven by spacer sequence TATA. Nucleic Acids Res 38:3827-33 |
| Huang, Shar-yin N; Crothers, Donald M (2008) The role of nucleotide cofactor binding in cooperativity and specificity of MutS recognition. J Mol Biol 384:31-47 |
| Zhang, Yongli; McEwen, Abbye E; Crothers, Donald M et al. (2006) Statistical-mechanical theory of DNA looping. Biophys J 90:1903-12 |
| Kuznetsov, Serguei V; Sugimura, Sawako; Vivas, Paula et al. (2006) Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF. Proc Natl Acad Sci U S A 103:18515-20 |
| Sugimura, Sawako; Crothers, Donald M (2006) Stepwise binding and bending of DNA by Escherichia coli integration host factor. Proc Natl Acad Sci U S A 103:18510-4 |
| Wickiser, J Kenneth; Winkler, Wade C; Breaker, Ronald R et al. (2005) The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol Cell 18:49-60 |
| Zhang, Yongli; Xi, Zhiqun; Hegde, Rashmi S et al. (2004) Predicting indirect readout effects in protein-DNA interactions. Proc Natl Acad Sci U S A 101:8337-41 |
| Bartoschek, Stefan; Buurman, Gerrit; Geierstanger, Bernhard H et al. (2003) Measurement and ab initio calculation of CSA/dipole-dipole cross-correlated relaxation provide insight into the mechanism of a H2-forming dehydrogenase. J Am Chem Soc 125:13308-9 |
| Barbic, Andrej; Zimmer, Daniel P; Crothers, Donald M (2003) Structural origins of adenine-tract bending. Proc Natl Acad Sci U S A 100:2369-73 |
| Zhang, Yongli; Crothers, Donald M (2003) Statistical mechanics of sequence-dependent circular DNA and its application for DNA cyclization. Biophys J 84:136-53 |
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