Structural Analysis of the Ribonuclease P Holoenzyme

提出的研究计划的重点是对细菌核糖核酸酶P （RNase P）全酶进行详细的结构分析。细菌RNase P从tRNA转录物中切割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，蛋白质是可缺性的。然而，RNase P蛋白对体内的生存能力至关重要，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rna单独反应有很大不同。虽然这种酶的催化RNA亚基已经被很好地表征，但人们对这种蛋白质片段如何介导其对酶促反应的多效作用知之甚少。具体解决的问题有：(a)定位RNase P RNA上的蛋白质结合位点，(b)鉴定功能所需的蛋白质结构域。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来研究。本研究将揭示RNase P蛋白及其在全酶中的功能；这将加强对RNA酶机制、RNA-蛋白以及RNA-RNA相互作用的理解。本研究计划的重点是对细菌核糖核酸酶P （RNase P）全酶进行详细的结构分析。人们对该酶的催化RNA亚基了解甚多，但对该蛋白质片段如何介导其对酶反应的多效作用知之甚少，而这些作用是体内功能所必需的。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. ***