We propose to describe the conformational transitions of paradigmatic nucleic acid complexes by using the results of solid state and solution NMR experiments to constrain long time scale computational simulations. In the previous funding period, we have demonstrated that solution and solid state NMR combined can cover motional processes that are not accessible to either technique separately, and have developed a formalism to extract the potential energy surface upon which dynamics occurs. We will apply these methods to describe at atomic resolution the sequence-specific conformational changes that occur in DNA as a methylase extrudes a base from the double helix. We will use the same methods in conjunction with real-time NMR methods to describe the conformational changes in a riboswitch at atomic resolution. We propose to study: I. The extrusion of a deoxycytidine from the double helix during DNA methylation - Using solid state and solution NMR methods together, we have observed unique motions within the target sequence for HhaI methylase. We hypothesize that these motions are specific to the GCGC repeat recognized by this enzyme and are important for base extrusion. II. The ligand-induced conformational changes in RNA - Riboswitches are genetic elements within the untranslated regions of mRNAs that regulate expression of certain enzymes. Direct binding of small metabolites to these RNAs alter their structures leading to up- or down-regulation of downstream open reading frames.

Public Health Relevance

The enzymatic methylation of DNA is essential for many biological processes, and abnormal patterns of DNA methylation is characteristic of many cancers. Riboswitches are RNA elements capable of directly regulating biosynthetic pathways in response to changing levels of particular metabolites, and are able to couple metabolite recognition with gene regulation in the complete absence of protein helpers. We seek to better understand the functional impact of structural flexibility in DNA methylation and in the functioning of riboswitches.

National Institute of Health (NIH)
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Mclaughlin, Alan Charles
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University of Washington
Schools of Arts and Sciences
United States
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