Structural Analysis of the Ribonuclease P Holoenzyme

9631039诺兰拟议的研究计划的重点是细菌核糖核酸酶P（RNase P）全酶的详细结构分析。 细菌RNase P从tRNA转录物切割5 '-前体序列。 它由一个大的（140 kd）RNA和一个小的（14 kd）蛋白质亚基组成。 在体外，RNA亚基是催化部分，而蛋白质是可降解的。 然而，RNase P蛋白对于体内生存力是必不可少的，并且体外全酶反应的动力学在盐需求、底物特异性和反应速率方面与单独的RNA反应显著不同。 虽然酶的催化RNA亚基的特点是，很少有人知道的蛋白质部分如何介导其多效性的酶促反应。 处理的具体问题有：（a）RNase P RNA上蛋白结合位点的定位，和（B）功能所需蛋白结构域的鉴定。这些问题将使用化学和酶足迹，交联分析，突变分析和动力学研究进行研究。 拟议的研究将阐明RNase P蛋白及其在全酶中的功能;这将增强对RNA酶机制以及RNA-蛋白质和RNA-RNA相互作用的理解。拟议的研究计划的重点是细菌核糖核酸酶P（RNase P）全酶的详细结构分析。 关于酶的催化RNA亚基，我们知道很多，但关于蛋白质部分如何介导其对酶促反应的多效性作用，这是体内功能所必需的，我们知之甚少。 所提出的实验将有助于理解蛋白质亚基如何与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. ***