Structural Analysis of the Ribonuclease P Holoenzyme

9631039诺兰拟议研究计划的重点是对细菌核糖核酸酶P(核糖核酸酶P)全酶进行详细的结构分析。细菌核糖核酸酶P从tRNA转录本中切割5‘-前体序列。它由一个大的(140kd)的RNA和一个小的(14kd)的蛋白质亚基组成。在体外，RNA亚单位是催化部分，蛋白质是必不可少的。然而，RNaseP蛋白是体内生存所必需的，体外全酶反应的动力学在盐需求、底物专一性和反应速度方面与单独的RNA反应有很大的不同。虽然该酶的催化RNA亚单位已被很好地描述，但人们对该蛋白质部分如何在酶反应中调节其多效性作用知之甚少。解决的具体问题包括：(A)RNaseP RNA上蛋白质结合位点的定位，以及(B)功能所需蛋白质结构域的鉴定。这些问题将通过化学和酶足迹、交联剂分析、突变分析和动力学研究来研究。这些研究将有助于阐明RNaseP蛋白及其在全酶中的功能；这将加深对RNA酶机制、RNA-蛋白质以及RNA-RNA相互作用的理解。拟议研究计划的重点是对细菌核糖核酸酶P(RNaseP)全酶进行详细的结构分析。人们对该酶的催化RNA亚基了解很多，但对该蛋白部分如何在体内发挥作用所必需的酶反应中介导其多效性效应却知之甚少。拟议的实验将有助于理解蛋白质亚单位如何与RNA相互作用，以及这种相互作用如何影响反应机制。***

9631039 Nolan The focus of the proposed research program is a detailed structural analysis of the bacterial ribonuclease P (RNase P) holoenzyme. Bacterial RNase P cleaves 5'-precursor sequences from tRNA transcripts. It consists of a large (140 kd) RNA and a small (14 kd) protein subunit. In vitro the RNA subunit is the catalytic moiety and the protein is dispensable. However, the RNase P protein is essential for viability in vivo, and the kinetics of the holoenzyme reaction in vitro differs dramatically from the RNA-alone reaction in salt requirement, substrate specificity, and reaction rate. While the catalytic RNA subunit of the enzyme is well characterized, little is known about how the protein moiety mediates its pleiotropic effects on the enzymatic reaction. Specific problems addressed are: (a) Localization of the protein binding site on RNase P RNA, and (b) Identification of protein domains required for function. These problems will be studied using chemical and enzymatic footprinting, crosslink analysis, mutational analysis, and kinetic studies. The proposed studies will shed light on the RNase P protein and its function in the holoenzyme; this will enhance understanding of the RNA enzyme mechanism, and RNA-protein as well as RNA-RNA interactions. %%% The focus of the proposed research program is a detailed structural analysis of the bacterial ribonuclease P (RNase P) holoenzyme. A great deal is known about the catalytic RNA subunit of the enzyme, but little is known about how the protein moiety mediates its pleiotropic effects on the enzymatic reaction, which are required for function in vivo. The proposed experiments will contribute to the understanding of how the protein subunit interacts with the RNA and how this interaction affects the reaction mechanism. ***