The central objective of this research program is to understand mechanisms of nucleic acid recognition in biological macromolecular assemblies. Emphasis is given to determining conformations and interactions of DNA in specific protein/DNA complexes that are relevant to gene regulation and chromosome organization. The methodology involves use of static and dynamic Raman spectroscopic probes of DNA structure and interaction.
The specific aims of the proposed research are as follows: (1) To determine sites of DNA base and backbone interactions and local and global DNA conformations in complexes of DNA with transcriptional activators and telomere binding proteins. (2) To extend and refine Raman spectral correlations for determining nucleic acid structures in nucleoprotein assemblies and condensed states of DNA, with emphasis on issues of DNA bending and folding. (3) To extend the time-resolution of Raman dynamic probes and the concentration range of quantitative Raman assays developed previously, thus enabling novel investigations of protein/DNA recognition.
These aims will be pursued using recently designed Raman approaches, including Raman microdialysis flow-cell technology, near-infrared/visible Raman spectrophotometry for ultrasensitive difference spectroscopy, Raman microspectroscopy, and ultraviolet-resonance Raman (UVRR) spectroscopy. Targeted for study are eukaryotic transcriptional activators (hSRY, LEF-1, bZIP protein GCN4) and prokaryotic factors (HU protein) bound to appropriate DNA sequences, telomere binding proteins in complexes with telomeric DNA, packaged viral chromosomes, and custom- designed DNA sequences with propensities for helix bending and folding. Interpretation of experimental results will be aided by theoretical normal mode analyses and by parallel studies of model nucleic acids for which high-resolution structures are available. The health-related impact of this program is derived from the fact that mechanisms of protein/DNA recognition will be elucidated for large biological assemblies, which are not amenable to investigation by alternative structural methods.
| Nemecek, Daniel; Stepanek, Josef; Thomas Jr, George J (2013) Raman spectroscopy of proteins and nucleoproteins. Curr Protoc Protein Sci Chapter 17:Unit17.8 |
| Tsuboi, Masamichi; Tsunoda, Masaru; Overman, Stacy A et al. (2010) A structural model for the single-stranded DNA genome of filamentous bacteriophage Pf1. Biochemistry 49:1737-43 |
| Tsuboi, Masamichi; Benevides, James M; Thomas Jr, George J (2009) Raman tensors and their application in structural studies of biological systems. Proc Jpn Acad Ser B Phys Biol Sci 85:83-97 |
| Benevides, James M; Danahy, Jessica; Kawakami, Jessica et al. (2008) Mechanisms of specific and nonspecific binding of architectural proteins in prokaryotic gene regulation. Biochemistry 47:3855-62 |
| Nemecek, Daniel; Gilcrease, Eddie B; Kang, Sebyung et al. (2007) Subunit conformations and assembly states of a DNA-translocating motor: the terminase of bacteriophage P22. J Mol Biol 374:817-36 |
| Benevides, J M; Li, T; Lu, X J et al. (2000) Protein-directed DNA structure II. Raman spectroscopy of a leucine zipper bZIP complex. Biochemistry 39:548-56 |
