HELICAL RNA, RNA:DNA HYBRIDS AND DNA-RNA JUNCTIONS

RNA双链体、RNA：DNA杂交双链体和DNA-RNA双链体连接 参与基因表达的许多重要步骤， 复制、转录和翻译控制。 相对 对这些生物学上重要的蛋白质的结构知之甚少， 双链体，主要是由于合成大的 大量的确定序列的RNA分子， 结构研究（NMR，X射线晶体学）。 我们有 绕过了这个瓶颈，最近合成了20-50 mg数量的具有确定序列的RNA链。 我们现在建议进行广泛的高分辨率核磁共振研究 对r：r双链体的溶液结构进行了研究， 同样的研究也将采用d：d结构进行 D：R杂合双链体和D-R-D：D-R-D嵌合双链体 包含A-B连接。 对结构的初步了解将从完整的 质子分配，化学位移变化， 耦合常数的变化。 更详细的结构 研究将涉及时间依赖性NOESY研究， 距离测量和从头生成三维 使用距离几何与这些距离一致的结构 算法 这些结果应该能让我们更清楚地了解 RNA的结构，控制DNA信息的转录 转化为RNA信息（反之亦然）， 复制叉处和DNA起始处的引物 复制的

RNA duplexes, RNA:DNA hybrid duplexes and DNA-RNA duplex junctions are involved in many important steps in gene expression including replication, transcription and translational control. Relatively little is known about the structure of these biologically important duplexes, largely due to the difficulty of synthesizing the large amounts of defined sequence RNA molecules required for detailed structural studies (NMR, x-ray crystallography). We have circumvented this bottleneck and have recently synthesized 20-50 mg quantities of RNA chains with defined sequences. We now propose to carry out extensive high-resolution NMR studies on the solution structure of r:r duplexes and to compare their structure to d:d duplexes of the same Studies will also be carried out on d:r hybrid duplexes and d-r-d:d-r-d chimeric duplexes containing A-B junctions. Initial insights into structure will be obtained from complete proton assignments, chemical shift changes, and interpretation of changes in coupling constants. More detailed later structural studies will involve time-dependent NOESY studies, interproton distance measurements and ab initio generation of three-dimensional structures consistent with these distances using distance geometry algorithms. The results should shed a great deal of light on the peculiarities of RNA structure, the control of transcription of DNA information into RNA information (and vice-versa) and the apparent need for RNA primers at replication forks and in the initiation of DNA replication.