Structural studies of RNase P

描述(申请人提供)：核糖核酸酶P(RNaseP)是一种核糖核蛋白复合体，负责处理细胞中许多不同的RNA分子。它存在于从细菌到人类的几乎所有生物体中，由一个必需的RNA亚基和一个或多个蛋白质亚基组成。RNA组分负责催化，因为它可以在没有蛋白质的情况下在体外处理RNA。在所有生物中，唯一共同的RNaseP功能是转移RNA(TRNA)的5‘端成熟。RNaseP是最早发现的催化RNA分子之一，它的研究对于我们理解RNA分子在催化中的作用是至关重要的。RNaseP是一种真正的多转位核酶，能识别其反式底物，也是仅有的两种通用核酶之一。对RNaseP结构和功能的了解有望为参与所有生物体共同的中央细胞过程的关键核酶提供重要和相关的信息，并加深我们对大RNA分子结构和功能的理解。这一建议涉及到RNaseP的结构和功能。在过去的几年里，我们在研究方面取得了实质性的进展，包括解决了Thermotoga maritima RNaseP全酶与成熟tRNA形成的复合体的结构。对全酶/tRNA复合体的结构研究表明，所有RNaseP核酶都有一个共同的基于RNA的RNA切割和识别机制，其中蛋白质组分通过准确定位5‘前导tRNA底物和接触P RNA结构的保守区来增加RNaseP的功能。结构还表明，RNaseP利用形状互补、特异的RNA-RNA接触和分子间碱基配对来有效识别其底物，P RNA和前tRNA都有助于协调两个对Pre-tRNA切割机制至关重要的催化重要金属离子。在下一阶段，我们建议继续和扩大我们对RNaseP的结构研究，这项建议的具体目标是：1)确定不同底物的RNaseP全酶复合体的三维结构，2)确定RNaseP全酶与前tRNA和过渡态类似物的复合体的结构，3)研究普遍保守的区域在RNaseP结构中的作用以及参与先导识别的重要氨基酸的作用。这项工作是基于分子生物学和生化方法的组合来生产和表征我们的工作所需的分子，并基于X射线结晶学来解决它们的原子结构。核糖核酸酶P的研究对健康相关研究具有重要意义。核糖核酸酶P是开发新的化疗药物的一个有希望的靶点，因为核糖核酸酶P的特异性可以改变，以产生降解靶RNA分子的分子。

DESCRIPTION (provided by applicant): Ribonuclease P (RNase P) is a ribonucleoprotein complex responsible for processing many different RNA molecules in the cell. It is found in almost all organisms, from bacteria to humans, and is composed of one essential RNA subunit and one or more protein subunits. The RNA component is responsible for catalysis as it can process RNA in vitro in the absence of protein. The only common RNase P function in all organisms is the 5' end maturation of transfer RNA (tRNA). RNase P 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 in our studies, including solving the structure of the complex formed by Thermotoga maritima RNase P holoenzyme and mature tRNA. The structural studies of the holoenzyme/tRNA complex show that all RNase P ribozymes share a common, RNA-based mechanism of RNA cleavage and recognition where the protein component increases RNase P functionality by accurately positioning the 5' leader pre-tRNA substrate and by contacting conserved regions of the P RNA structure. The structure also shows that RNase P utilizes shape complementarity, specific RNA-RNA contacts, and intermolecular base pairing to recognize its substrate efficiently and that both P RNA and the pre-tRNA help to coordinate two catalytically important metal ions essential for the mechanism of pre-tRNA cleavage. For the next period we propose to continue and expand our structural studies of RNase P. The specific aims for this proposal are: 1) to determine the three dimensional structure of complexes of RNase P holoenzyme with different substrates, 2) to determine the structure of complexes of RNase P holoenzyme with pre-tRNA and transition state analogues and, 3) to study the role of the universally conserved regions in the structure of RNase P and also the role of important amino acids involved in leader recognition. 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. The work on RNase P has important implications for health related studies. RNase P is a promising target for the development of new chemotherapeutics as the specificity of RNase P can be altered to create molecules that degrade target RNA molecules.