STRUCTURE/FUNCTION OF RIBONUCLEASE P

这项研究计划的主要目标是了解基础知识 RNA的结构和催化功能方面。核糖核糖核酸亚基 细菌核糖核酸酶P(RNaseP)，催化特定的 前-tRNA的切割，之所以被选为研究对象，是因为它代表了 催化RNA分子的基本、广泛和保守的呼唤 (核酶)。研究计划的具体内容是：1.两 将采用互补的方法来确定准确的 核酶上的残基参与底物结合和催化。 分子间交联剂，使用随机和定点两种方法 修饰的底物，将用于测定核糖核酸酶 与底物上的已知接触部位并列。残余物 化学修饰干扰结合或催化 通过使用一系列新的核苷酸类似物在 修饰-干扰实验。这些结果预计将 建议特定的结构元素，包括分子间 联系人。2.含-位核酶的反应动力学 将检查特定的官能团修改以进行测试 对具体目标1的分析表明了潜在的相互作用。 稳态、单次周转和绑定动力学将在 以区分对催化和结合的影响。一个 利用自切割核酶-底物偶联物的新策略 将用于简化突变或突变的催化速率测定 修饰的核酶。3.核酶内的第三次接触将是 通过分析分离的RNase P RNA结构域的关联来确定 在试管中。为了便于检测结合，前tRNA序列将 融合到单独的域和域间交互将是 用分子间裂解法检测。4.核酶的动力学 包括折叠和底物诱导的构象变化的结构 将使用光亲和交联法进行分析。分析 将揭示次生和第三系的形成顺序 并将包括对化学因素的评估 它们会影响它们的形成。结构的识别 游离核酶与核酶-底物复合体的区别 将有助于定义伴随着多次更替的重新安排 核糖核酸酶P反应。 该研究计划提供的数据将有助于揭示 RNA功能方面；包括结构、RNA-RNA的性质 相互作用和RNA介导的催化作用。更好地理解 这些问题将有助于提高我们对 基于RNA的治疗工程的指导原则，提供 在疾病治疗方面新方向的巨大潜力。

The broad goal of this research program is to understand fundamental aspects of RNA structure and catalytic function. The RNA subunit of bacterial ribonuclease P (Rnase P), which catalyzes the specific cleavage of pre-tRNA, has been chosen for study because it represents an essential, widespread and conserved call of catalytic RNA molecules (ribozymes). The specific elements of the research program are: 1. Two complementary approaches will be employed to pinpoint the precise residues on the ribozyme involved in substrate-binding and catalysis. Intermolecular crosslinking, using both randomly and site-specifically modified substrates, will be used to determine riboyme nucleotides juxtaposed to known contact sites on the substrate. Residues where chemical modification interferes with binding or catalysis will be identified by employing a series of novel nucleotide analogs in modification-interference experiments. These results are expected to suggest specific elements of structure, including intermolecular contacts. 2. The reaction kinetics of ribozymes containing site- specific functional group modifications will be examined to test potential interactions indicated by the analyses of Specific Aim 1. Steady-state, single-turnover and binding kinetics will be measured in order to differentiate between effects on catalysis and binding. A novel strategy employing self-cleaving ribozyme-substrate conjugates will be used to simplify determination of catalytic rate of mutant or modified ribozymes. 3. Tertiary contacts within the ribozyme will be determined by analyzing the association of isolated Rnase P RNA domains in vitro. To facilitate detection of binding, pre-tRNA sequences will be fused to individual domains and inter-domain interactions will be assayed by intermolecular cleavage. 4. The dynamics of ribozyme structure including folding and substrate-induced conformational changes will be analyzed using a photoaffinity crosslinking approach. Analysis of folding will reveal the order of formation of secondary and tertiary interactions and will include an assessment of the chemical factors which influence their formation. Identification of structural differences between the free ribozyme and ribozyme-substrate complex will help define rearrangements that accompany the multiple turnover RNase P reaction. The data provided by the research program will shed light on fundamental aspects of RNA function; including structure, the nature of RNA-RNA interactions, and RNA-mediated catalysis. A better comprehension of these issues will lead to improvement in our understanding of the guiding principals for engineering RNA-based therapeutics which offer enormous potential for new directions in treatment of disease.