Dynamic Aspects of Nucleic Acid-Protein Recognition

描述（由申请人提供）：我们建议通过使用固态和溶液NMR实验的结果来描述范例核酸复合物的构象转变，以约束长时间尺度的计算模拟。在上一个资助期，我们已经证明，溶液和固态NMR相结合，可以覆盖运动过程，这是无法单独访问的技术，并已开发出一种形式主义，以提取势能面上发生的动态。我们将应用这些方法来描述在原子分辨率的序列特异性的构象变化，发生在DNA中的甲基化酶挤出从双螺旋的碱基。我们将使用相同的方法结合实时核磁共振方法来描述原子分辨率的核糖开关的构象变化。我们建议研究：一。DNA甲基化过程中脱氧胞苷从双螺旋中的挤出-同时使用固态和溶液NMR方法，我们观察到HhaI甲基化酶靶序列内的独特运动。我们推测，这些运动是特定的GCGC重复识别这种酶，是重要的基础挤出。二.核糖开关是位于mRNA非翻译区的遗传元件，它调节某些酶的表达。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的酶促甲基化对于许多生物学过程是必不可少的，并且DNA甲基化的异常模式是许多癌症的特征。核糖开关是能够响应于特定代谢物水平的变化而直接调节生物合成途径的RNA元件，并且能够在完全不存在蛋白质辅助物的情况下将代谢物识别与基因调控偶联。我们试图更好地了解DNA甲基化和核糖开关功能的结构灵活性的功能影响。

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.