PHYSICAL STUDIES OF RNA STRUCTURAL MOTIFS

许多不同的RNA分子参与细胞中的基因表达。 在 在过去的几年里，一些重要的大RNA的二级结构， 已经通过序列比较方法推导出，并且越来越清楚 某些结构基序对这些功能很重要， RNA。 这项提案的目的是研究三维 这些结构基序的形成的结构和热力学。 合成RNA将被设计成在分子中复制这些结构 小到足以进行详细的物理研究。 待研究的特定基序是单碱基凸起（对于 蛋白质识别）;假结，或发夹环之间的碱基配对 （可能参与RNA的结构重排）;和螺旋 含有大量的非典型碱基对。 使用体外 通过纯化的T7聚合酶转录克隆的合成DNA，20-30个碱基 含有这些结构的RNA分子将被合成。 10 必要时可制备毫克量。 我们的几种产品 将进行物理测量以确定结构， 这些RNA的热力学：相关RNA的解链温度， 嵌入剂- RNA或RNA-RNA相互作用常数和质子NMR 谱 此外，这些模型RNA的化学修饰研究 将提供一种方法来比较它们与大RNA的构象。

Many different RNA molecules are involved in gene expression in cells. In the last few years the secondary structures of several important large RNAs have been deducedd by sequence comparison methods, and it is becoming clear that certain structural motifs are important for the function of these RNAs. The object of this proposal is to study the three dimensional structures and thermodynamics of formation of these structural motifs. Synthetic RNAs will be designed to reproduce these structures in molecules small enough to be amenable to detailed physical studies. The specific motifs to be studied are single base bulges (important for protein recognition); pseudoknots, or base pairing between hairpin loops (probably involved in structural rearrangements of RNAs); and helices containing large proportions of non-canonical base pairs. Using in vitro transcription of cloned synthetic DNA by purified T7 polymerase, 20-30 base RNA molecules containing these structures will be synthesized. 10 milligram amounts can be prepared when necessary. Several kinds ouf physical measurements will be made to determine the structure and thermodynamics of these RNAs: melting temperatures of related RNAs, binding constants for intercalator - RNA or RNA - RNA interctions, and proton NMR spectra. In addition, chemical modification studies of these model RNAs will provide a way to compare their conformations with those of large RNAs.