STRUCTURE/FUNCTION OF RIBONUCLEASE P

这项研究计划的主要目标是了解基本的 RNA的结构和催化功能。 RNA亚基 细菌核糖核酸酶P（RNase P），催化特异性 选择前tRNA切割进行研究，因为它代表了 一种重要的、广泛的和保守的催化RNA分子 （核酶）。 研究方案的具体内容是：1. 两 将采用互补的方法来确定 核酶上参与底物结合和催化的残基。 分子间交联，使用随机和位点特异性 修饰的底物，将用于确定核糖核酸 与衬底上的已知接触位置并置。 的残 化学修饰会干扰结合或催化作用， 通过使用一系列新的核苷酸类似物鉴定， 修饰干扰实验 这些结果预计将 提出了结构的具体要素，包括分子间 联系人. 2.研究了含位点的核酶的反应动力学， 将检查特定的官能团修饰以测试 具体目标1的分析表明了潜在的相互作用。 稳态、单转换和结合动力学将在 以区分对催化和结合的影响。 一 使用自切割核酶-底物缀合物的新策略 将用于简化突变体或 修饰的核酶。 3. 核酶内的三级接触将是 通过分析分离的RNase P RNA结构域的结合来确定 体外为了便于检测结合，前tRNA序列将 融合到各个域，域间的交互将 通过分子间裂解测定。 4. 核酶的动力学 结构包括折叠和底物诱导的构象变化 将使用光亲和交联方法进行分析。 分析 将揭示二级和三级构造的形成顺序 并将包括对化学因素的评估 影响其形成。 识别结构 游离核酶和核酶-底物复合物之间的差异 将有助于定义伴随着多次周转的重新安排， RNase P反应。 该研究项目提供的数据将揭示基本的 RNA功能的各个方面;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 这些问题将有助于我们更好地了解 设计基于RNA的治疗方法的指导原则， 在疾病治疗的新方向上具有巨大的潜力。

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.