RUI: Structrual Analysis of RNase P, An RNA Catalyst

随着RNA分子在中心链的二级反应中作为催化剂的发现，RNA的动态能力已得到充分认识。RNase P RNA对其底物（前体tRNA）的内溶活性是第二种被描述的这种活性。建议继续对RNase P的结构进行研究，以进一步明确RNase P的催化机制和胞内结构，进一步扩大对该RNA催化剂系统发育的认识，并开始寻找其他生理上重要的小RNA。RNase P RNA的结构分析将利用来自几种微生物的RNase P RNA的比较测序以及在不同温度和离子条件下的结构化学和酶探针。RNase P的系统发育将通过使用合成前体tRNA作为底物检测耐辐射球菌、玫瑰微球菌、产孢梭菌、日光盐杆菌和嗜酸热原体来探索。RNase P的细胞内构象将通过在保持大分子相互作用的条件下分离活性来检查，或者通过询问纯化的RNase P RNA结合的蛋白质来检查。其他分离的小rna将被用于测试进化保护（因此具有生理重要性）。最近发现RNA在其自身成熟过程中催化某些反应。这与以前认为只有蛋白质才能作为催化剂（酶）的观念相反。这也导致了RNA分子是第一个自我复制的生命单位的明显可能性。这项提议是为了研究这些RNA颗粒的结构，并确定它们是否存在于地球上仍然存在的最古老的生命形式——古细菌中。

The dynamic capabilities of RNA have been fully realized with the discovery that RNA molecules can perform as catalysts in secondary reactions of the central dogma. The endonucleolytic activity of RNase P RNA on its substrate, precursor tRNA, was the second such activity to be described. It is proposed to continue the investigation on the structure of RNase P in order to further define the mechanism of catalysis and the intracellular structure of RNase P. Further it is proposed to extend the understanding of the phylogenetics of this RNA catalyst as well as to begin a search for other physiologically important small RNAs. The structural analysis of RNase P RNA will make use of comparative sequencing of RNase P RNAs from several microorganisms as well as chemical and enzymatic probes of structure at various temperatures and ionic conditions. The phylogenetics of RNase P will be explored by examining Deinococcus radiodurans, Micrococcus roseus, Clostridium sporogenes, Halobacter solanarium, and Thermoplasma acidophilum using a synthetic precursor tRNA as substrate. The intracellular conformation of the RNase P will be examined by the isolation of the activity under conditions that preserve macromolecular interactions and, alternatively, by asking what proteins bind to purified RNase P RNA. Other small RNAs isolated will be tested for evolutionary conservation (hence physiological importance). RNA has recently been found to catalyze certain reactions in its own maturation process. This is contrary to the previously held concept that only proteins could serve as catalysts (enzymes). It also leads to the obvious possibility that RNA molecules were the first self-replicating units of life. This proposal is to investigate the structure of these RNA particles and to determine whether they are present in the most ancient forms of life still present on the planet, the archaeacteria.