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STRUCTURE/FUNCTION ANALYSIS OF RNASE P, AN RNA CATALYST

RNA 催化剂 RNA酶 P 的结构/功能分析

基本信息

批准号：
3438478
负责人：
TERRY L MARSH
金额：
$ 7.5万
依托单位：
HAMILTON COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-09-01 至 1988-08-31
项目状态：
已结题

项目摘要

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.

RNA分子的潜在动力学方面强调了 RNase 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。我们我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RNase P在整个细胞中的普遍存在。三国本研究提案针对这些缺点，建议将对生物学上重要的小RNA的搜索扩展到其他的物种和王国以下列方式。 1.继续分析RNase P RNA结构。一个最可靠的核酸结构分析的方法是比较序列法分析. 我们打算从几种不同的细胞中分离RNase P RNA并进行测序，代表性生物作为确定二级结构的手段。 2.在亲缘关系较远的属和界中寻找RNase P。的我们特别感兴趣的是RNase P活性的性质，古细菌，一种古老的生物谱系。我们计划寻找 RNase P在Halobacterium volcanii，H.盐藻，可能还有硫化叶菌太阳神 3.评估活动的其他细胞成分的影响和B的构象。在分离RNase P的过程中，来自B的蛋白质。枯草芽孢杆菌中，14个蛋白质被纯化至接近均一。发现这些蛋白质中至少有一种抑制RNase P活性。我们我建议进一步研究这些蛋白质的抑制或刺激作用，活性以及测定它们对RNase P RNA的亲和力（如果有的话）， tRNA。 4.在一定条件下检查tRNA底物的结构最佳的RNase P活性。当然，切割，因此切割可能严重依赖于 tRNA底物的构象。我们计划对此进行调查使用分光光度计和酶分析前体的可能性和成熟的底物结构。 5.我们将研究代表性的小RNA的星座，微生物包括B。枯草芽孢杆菌（B. subtilis）、B.短球菌，屎链球菌，和 H.火山 RNA将通过色谱柱组合分离，层析和凝胶电泳，通过Southern分析表征并尽可能地进行排序。