STRUCTURE/FUNCTION ANALYSIS OF RNASE P, AN RNA CATALYST

RNA分子的潜在动态方面被强调为 核糖核酸酶P-RNA对其底物--前体tRNA的核酸内切活性 RNase P RNA是第二个被描述为具有催化活性的RNA。我们 对这种分子的结构或构型知之甚少 在细胞内，甚至更少关于RNaseP在整个细胞中的普遍存在 三个王国。这项研究提案解决了这些不足，并 建议将对具有生物重要性的小RNA的搜索范围扩大到 其他物种和王国以下列方式。 1.继续分析核糖核酸酶P-RNA结构。其中一个最可靠的 核酸结构分析的方法是比较序列 分析。我们打算从几个物种中分离和测序RNaseP RNA 具有代表性的生物体作为确定二级结构的手段。 2.寻找远缘关系属和科属的RNaseP。的 我们特别感兴趣的是核糖核酸酶P活性的性质 古细菌，一种古老的有机体谱系。我们计划去寻找 火山嗜盐杆菌、盐生嗜盐杆菌和硫杆菌中的核糖核酸酶P 索法里克斯。 3.评估活动的其他细胞成分的影响 和枯草杆菌核糖核酸酶P分离过程中的构象。 从枯草杆菌中分离纯化了14种蛋白质，得到了接近均一的蛋白质。 至少有一种蛋白质被发现抑制RNaseP的活性。我们 建议进一步研究这些蛋白质的抑制性或刺激性 活性以及确定它们对RNaseP RNA的亲和力(如果有的话)和 TRNA。 4.检测条件下tRNA底物的结构 最适合核糖核酸酶P活性。当然，这是底物 卵裂，因此卵裂可能严重依赖于 TRNA底物的构象。我们计划对此进行调查 对前体进行分光光度和酶分析的可能性 在最佳的RNA加工条件下形成成熟的底物结构。 5.我们将研究具有代表性的小RNA星座 微生物包括枯草杆菌、短杆菌、粪链霉菌和 丛枝杆菌属。RNAs将通过组合柱子来分离 用Southern分析表征的层析和凝胶电泳法 并在可能的情况下进行测序。

The potential dynamic aspect of RNA molecules is underscored by the endonucleolytic activity of RNase P RNA on its substrate, precursor tRNA. RNase P RNA was the second RNA to be described with catalytic activity. We know little about the structure of this molecule or its configuration within the cell, and even less about the ubiquity of RNase P throughout the three kingdoms. This research proposal addresses these shortcomings and proposes to extend the search for biologically important small RNAs to other species and kingdoms in the following ways. 1. Continued analysis of RNase P RNA structure. One of the most reliable methods of structure analysis of nucleic acids is comparative sequence analysis. We intend to isolate and sequence RNase P RNA from several representative organisms as a means of determining secondary structure. 2. Search for RNase P in distantly related genera and kingdoms. Of particular interest to us is the nature of RNase P activity in the Archaebacteria, an ancient lineage of organisms. We plan to search for RNase P in Halobacterium volcanii, H. salinarium, and possibly Sulfolobus solfataricus. 3. Assessment of the influence of other cellular components of the activity and conformation of B. subtilis RNase P. During the isolation of RNase P protein from B. subtilis, 14 proteins were purified to near homogeneity. At lease one of these proteins was found to inhibit RNase P activity. We propose to further examine these proteins for inhibitory or stimulatory activity as well as determine their affinity, if any, for RNase P RNA and tRNA. 4. Examination of the structure of the tRNA substrate under conditions optimal for RNase P activity. It is of course the substrate that is cleaved and therefore cleavage may be critically dependent on the conformation of the tRNA substrate. We plan to investigate this possibility using spectrophotometric and enzymatic analysis of precursor and mature substrate structure under optimal RNA processing conditions. 5. We will examine the constellation of small RNAs within representative microorganisms including B. subtilis, B. brevis, Streptoccus faecium, and H. vulcanii. The RNAs will be isolated by a combination of column chromatography and gel electrophoresis, characterized by Southern analysis and sequenced when possible.