Dynamic Aspects of Nucleic Acid-Protein Recognition

描述（由申请人提供）：我们建议用固态和溶液核磁共振实验的结果来描述聚合核酸复合物的构象转变，以约束长时间尺度的计算模拟。在之前的资助期内，我们已经证明了溶液和固态核磁共振结合可以覆盖单独使用任何一种技术都无法访问的运动过程，并且已经开发了一种形式来提取动态发生的势能表面。我们将应用这些方法在原子分辨率上描述当甲基化酶从双螺旋中挤出碱基时发生在DNA中的序列特异性构象变化。我们将使用相同的方法结合实时核磁共振方法来描述核开关在原子分辨率上的构象变化。我们建议研究：1 . DNA甲基化过程中双螺旋脱氧胞苷的挤压-使用固态和溶液核磁共振方法一起，我们观察了HhaI甲基化酶目标序列内的独特运动。我们假设这些运动是特异性的GCGC重复识别的酶和重要的碱挤出。2。配体诱导的RNA -核糖开关的构象变化是mrna非翻译区域内调节某些酶表达的遗传元件。小代谢物与这些rna的直接结合改变了它们的结构，导致下游开放阅读框的上调或下调。公共卫生相关性：DNA的酶甲基化对许多生物过程至关重要，DNA甲基化的异常模式是许多癌症的特征。核糖开关是一种RNA元件，能够直接调节生物合成途径，以响应特定代谢物水平的变化，并且能够在完全没有蛋白质辅助的情况下将代谢物识别与基因调控结合起来。我们试图更好地理解结构灵活性对DNA甲基化和核开关功能的功能影响。

DESCRIPTION (provided by applicant): 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.