Structural studies of the RNA component of RNase P

描述(申请人提供)：转移RNA是作为前体分子产生的，需要在其3‘和5’端进行处理。核糖核酸酶RNase是tRNA P或P 5‘端的内切酶，只负责切割前体并导致tRNA成熟。它含有一种RNA和一种蛋白质成分，在所有生物体中都已被发现。它是最早被发现的催化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至关重要。核糖核酶P是一种真正的多转位核酶，也是三个生命王国中仅存的两种核酶之一。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。本研究涉及细菌核糖核酸酶P的结构和功能，细菌核糖核酸酶P的RNA成分由两个结构域组成：特异性结构域和催化结构域。最近，我们解决了枯草杆菌核糖核酸酶P的特异域的结构，现在我们建议扩大我们的研究范围，以更全面地表征枯草杆菌核糖核酸酶P的特异域，研究其他细菌的核糖核酸酶P的结构，并研究完整的全酶的结构。本研究的具体目的是：1)确定嗜热杆菌核糖核酸酶P专一性结构域的结构；2)获得枯草杆菌核糖核酸酶P专一性结构的较高分辨率信息；3)研究枯草杆菌核糖核酸酶P与金属离子及其底物的相互作用；4)结晶并解析完整的枯草杆菌核糖核酸酶P全酶结构。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工作所需的分子，并基于X射线结晶学来解决它们的结构。

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 RNase is the endonuclease for the 5' end of tRNA P, or P, only responsible processing by cleaving a precursor and leading to tRNA maturation. It contains an RNA and a protein component and has been identified in all organisms. It is one of the first catalytic RNA molecules identified 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 and one of only two ribozymes conserved in all three kingdoms of life. 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 bacterial RNase P. The RNA component of bacterial RNase P is made of two domains: a specificity and a catalytic domain. Recently, we solved the structure of the specificity domain of B. subtilis RNase P and we are now proposing to expand our studies to characterize more fully the specificity domain of B. subtilis RNase P, to study the structure of other bacterial RNase P, and to study the structure of the intact holoenzyme. The specific aims for this proposal are: 1) to determine the structure of the T. thermophilus RNase P specificity domain, 2) to obtain higher resolution information on the B. subtilis RNase P specificity domain, 3) to study the interactions of B. subtilis RNase P with metal ions and with its substrate, and 4) to crystallize and solve the structure of the intact B. subtilis RNase P holoenzyme. 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 structures.