STRUCTURE AND FUNCTION OF 5S RNA FLEXIBILITY

核糖体RNA不再被认为是纯粹的结构成分 但被认为可能在 在蛋白质合成中的作用 对其作用的重新评估一直是 部分原因是RNA研究方法的进步 结构-功能相关性，也部分由 发现其他具有催化活性的RNA分子，如 RNase P.如何“简单”的大分子，如RNA，只由 四个单体，可以催化功能是一个显着的 化学生物学问题。 这种RNA序列的一个关键特征是 被认为涉及构象柔性。 更彻底的 了解构象柔性的结构基础 将为分子生物学的所有领域提供信息， 通过扩展，这些医学科学领域的分子， 科学直接冲击。 在拟议的项目中， 关于构象的功能重要性的假说 5S核糖体RNA的HelixII-HelixIII区域的柔性将 得到考验 实验方法采用以下组合： 遗传、生物化学和物理化学方法来回答这个问题。 以下问题：（1）构象潜力 5S RNA的生物学意义？ (2)5S RNA在 事实上构象灵活的意义上说，螺旋II-III 区域可以在两个（或更多个）二级结构之间切换， 解决方案？ (3)如果转换发生，能量学和 构象变化的动力学，以及这三个- 所涉及的国家的维度结构？ 功能研究 第一个问题（1），首先要解决的是 证明该项目的生物学意义， 问题（2）和（3），其中涉及更大的 资源承诺，特别是密集使用高 场核磁共振波谱学 突变将在克隆的 编码来自大肠杆菌的真细菌5S RNA的基因， 改变RNA的一级结构， 改变其构象柔性。 改变的5S RNA 通过突变基因的体外或体内转录制备 并进行生物学功能测试，包括与核糖体结合， 蛋白L25和L18，并掺入50 S核糖体 亚单位。 成功地重组了含有 将测试改变的RNA的tRNA结合和蛋白质合成。 将采用高场二维核磁共振技术， 表征溶液结构和构象动力学 的分子。 建议的结构研究将在 （1）设计用于模拟 在HSP 73 III（2）RNA序列中的两个碱基凸起结构， 螺旋II和III的整个转换区（正常序列如 以及改变的、构象上“锁定”的序列）;和（3）所述序列中的 带有相同突变的完整5S RNA分子。

Ribosomal RNA is no longer considered a purely structural component of ribosomes, but thought perhaps to play the central catalytic role in protein synthesis. This reassessment of its role has been prompted in part by methodological advances in studies of RNA structure-function correlations, and also in part by the discoveries of other catalytically active RNA molecules, such as RNase P. How "simple" macromolecules like RNA, consisting only of four monomers, can function catalytically is a significant chemical-biological question. A key feature of such RNA sequences is thought to involve conformational flexibility. A more thorough understanding of the structural basis of conformational flexibility in nucleic acids will inform all areas of molecular biology and, by extension, those areas of medical science upon which molecular sciences directly impinge. In the proposed project, a specific hypothesis regarding the functional importance of conformational flexibility in the HelixII-HelixIII region of 5S ribosomal RNA will be tested. The experimental approach employs a combination of genetic, biochemical, and physical-chemical methods to answer the following questions: (1) is the potential for conformational flexibility in 5S RNA biologically significant? (2) Is 5S RNA in fact conformationally flexible in the sense that the HelixII-III region can switch between two (or more) secondary structures in solution? (3) If switching occurs, what are the energetics and dynamics of the conformational change and what are the three- dimensional structures of the states involved? Functional studies aimed at answering Question (1) will be carried out first to demonstrate the biological significance of the project, before proceeding with questions (2) and (3), which involve a greater commitment of resources, in particular the intensive use of high- field NMR spectroscopy. Mutations will be constructed in a cloned gene coding for the eubacterial 5S RNA from E.coli, which will alter the RNA's primary structure in ways that are expected to alter its conformational flexibility. Altered 5S RNA's will be prepared by in vitro or in vivo transcription of the mutated genes and tested for biological function, including binding to ribosomal proteins L25 and L18, and incorporation into 50S ribosomal subunits. Successfully reconstituted ribosomes containing the altered RNAs will be tested for tRNA binding and protein synthesis. High-field, two-dimensional NMR techniques will be undertaken to characterize the solution structure(s) and conformational dynamics of the molecule. The proposed structural studies will be carried out on (1) short RNA sequences which are designed to model the two-base bulge structure in Helix III (2) RNA sequences which model the entire switch region of Helices II and III (normal sequence as well as altered, conformationally "locked" sequences); and (3) the intact 5S RNA molecules bearing these same mutations.