Structural studies of RNase P

描述（由申请人提供）：转移RNA作为前体分子产生，需要在其3'和5'末端进行加工。核糖核酸酶P或RNase P是负责通过切割前体并导致tRNA成熟来加工tRNA的5'末端的唯一内切核酸酶。它由一个大的RNA分子和至少一种蛋白质组成，在所有生物体中都有发现。它是最早发现的催化RNA分子之一，它的研究对我们理解RNA分子在催化中的作用至关重要。核糖核酸酶P是一种真正的多转换核酶，它以反式识别其底物，是仅有的两种通用核酶之一。核糖核酸酶P的结构和功能的知识，承诺提供重要的和相关的信息，一个关键的核酶参与一个中央的细胞过程中常见的所有生物体，也进一步了解大RNA分子的结构和功能。该提案涉及RNase P的结构和功能。在过去的几年里，我们取得了实质性的进展，这一结构为RNase P的RNA组分提供了重要的结构信息，使我们能够对这种通用核酶的功能进行关键性的观察，并将结构与现有的丰富的生化数据。在下一个资助期内，我们建议继续并扩大我们对RNase P的研究。这项建议的具体目标是：1）确定RNase P全酶与tRNA复合物的三维结构，2）扩展我们对T. 3）研究RNase P结构中普遍保守区域的作用及其对RNA识别和切割的影响。这项工作是基于分子生物学和生物化学方法的结合，以产生和表征我们工作所需的分子，以及X射线晶体学来解决它们的原子结构。

DESCRIPTION (provided by applicant): Transfer RNA is produced as a precursor molecule that needs to be processed both at its 3' and 5' ends. Ribonuclease P, or RNase P, is the only endonuclease responsible for processing the 5' end of tRNA by cleaving a precursor and leading to tRNA maturation. It is composed of a large RNA molecule and at least one protein and it has been identified in all organisms. It was one of the first catalytic RNA molecules discovered and its study has been pivotal to our understanding of the role of RNA molecules in catalysis. RNase P is a true multi-turnover ribozyme that recognizes its substrate in trans and one of only two universal ribozymes. The knowledge of the structure and function of RNase P promises to provide important and relevant information on a key ribozyme involved in a central cellular process common to all organisms and also to further our understanding of the structure and function of large RNA molecules. This proposal is concerned with the structure and function of RNase P. In the past few years we have made substantial progress, including solving the structure of the intact RNA component of Thermotoga maritima RNase P. This structure provided important structural information on the RNA component of RNase P and allowed us to make crucial observations regarding the function of this universal ribozyme and relate the structure to the wealth of existing biochemical data. For the next funding period we propose to continue and expand our studies of RNase P. The specific aims for this proposal are: 1) to determine the three dimensional structure of a complex of the RNase P holoenzyme with tRNA, 2) to extend our structural studies of the RNA component of T. maritima RNase P to higher resolution and, 3) to study the role of the universally conserved regions in the structure of RNase P and the implications for RNA recognition and cleavage. The work is based on a combination of molecular biology and biochemical methods to produce and characterize the molecules that we require for our work, and X-ray crystallography to solve their atomic structures.