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

RNA双链、RNA：DNA杂合双链和DNA-RNA双链连接 参与了基因表达的许多重要步骤，包括 复制、转录和翻译控制。相对的 人们对这些具有重要生物学意义的分子的结构知之甚少 双链，很大程度上是由于合成大型 详细说明所需的已定义序列RNA分子的数量 结构研究(核磁共振、X射线结晶学)。我们有 绕过了这一瓶颈，最近合成了20-50 Mg数量的具有定义序列的RNA链。 我们现在提议进行广泛的高分辨率核磁共振研究 关于R：R双链的溶液结构及其比较 还将携带相同研究的结构到D：D的双面 关于d：r混合双链和d-r-d：d-r-d嵌合双链 包含A-B连接点。 对结构的初步了解将从Complete中获得 质子赋值、化学位移变化和对 耦合常数的变化。更详细的后续结构 研究将涉及依赖时间的NOESY研究，质子间 三维空间的距离测量和从头算生成 使用距离几何与这些距离保持一致的结构 算法。 这一结果应该能很好地揭示这些奇特之处 对RNA结构、DNA信息转录的控制 转化为RNA信息(反之亦然)以及对RNA的明显需求 复制分叉和DNA起始中的引物 复制。

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.