EUKARYOTIC TRNA MATURATION

核糖核酸酶P(RNase P)裂解前tRNA，得到成熟的5‘ 终极迷你。细菌、古生菌和真核生物的核糖核酸酶P全酶 已被证明含有相似大小的基本RNA亚基和 结构，尽管在大小、数量 以及蛋白质组分的贡献。核糖核酸酶P在 已知的核溶解RNA是指一个单一的酶识别一个大的 通过三次接触的基片数量，而不是沃森-克里克 碱基配对。拟议研究的目的是调查 酿酒酵母核糖核酸酶P是如何识别和切割其 底物和这种全酶如何与核加工相互作用 TRNA初级转录本的途径。 在之前的一段时间里，这个实验室提出了二级和三级 酵母核核糖核酸酶P-RNA的结构模型的功能 对RNA模型中高度保守的序列和结构进行了测试 定向诱变及其产物全酶的鉴定 在体内组装。为了帮助生化鉴定，我们提纯了 核糖核酸酶，并鉴定出一个复杂的亚基结构，包括 在与RNA亚单位紧密相关的九种蛋白质中。所有子单位 被证明是生命和体内RNaseP功能所必需的，以及 九个蛋白质亚基中的八个也与RNaseMRP相关， 一种与Re-核糖体RNA加工有关的内切酶 核仁。这种与RNaseMRP的密切关系促使我们检查 核糖核酸酶P是否也可能是核仁，导致发现 包括RNaseP在内的大部分前tRNA加工途径都位于 在核仁中。未来的工作将基于这样的假设： 有限数量的RNaseP蛋白亚基直接参与 与RNA亚单位在前tRNA裂解，而大多数 酶蛋白含量赋予复合核所需的功能 环境。在这项提案中，几个具体问题将是 关于酵母核糖核酸酶的结构和功能 P[1]核糖核酸酶P底物如何被RNA和蛋白质识别 这种复杂的全酶的组成成分？[2] 全酶结合在一起才能实现这一功能？[3]还有什么其他的？ 蛋白质亚基在细胞生理功能中的作用 核tRNA加工途径中的全酶？

Ribonuclease P (RNase P) cleaves pre-tRNAs to give the mature 5' termini. RNase P holoenzymes from Bacteria, Archaea and Eucarya have been shown to contain essential RNA subunits of similar size and structure, although there are pronounced differences in the size, number and contribution of the protein components. RNase P is unusual among the known nucleolytic RNAs in that a single enzyme recognizes a large number of substrates through tertiary contacts, rather than Watson-Crick base pairing. The purpose of the proposed research is to investigate how Saccharomyces cerevisiae nuclear RNase P recognizes and cleaves its substrates and how this holoenzyme interacts with the nuclear processing pathway for tRNA primary transcripts. In the previous period, this laboratory proposed secondary and tertiary structure models for the yeast nuclear RNase P RNA. The function of highly conserved sequences and structures in the RNA models were tested by directed mutagenesis and characterization of the resulting holoenzyme assembled in vivo. To aid in biochemical characterization, we purified the nuclear RNase and identified a complex subunit structure consisting of nine proteins tightly associated with the RNA subunit. All subunits are shown to be essential for life and for RNase P function in vivo, and eight of the nine protein subunits are also associated with RNase MRP, a related endonuclease involved in -re-ribosomal RNA processing in nucleoli. This close relationship to RNase MRP caused us to examine whether RNase P might also be nucleolar, leading to the discovery that much of the pre-tRNA processing pathway, including RNase P, is located in the nucleolus. Future work will proceed from the hypothesis that a limited number of the protein subunits of RNase P participate directly with the RNA subunit in pre-tRNA cleavage, while the majority of the enzyme protein content confers functions required in the complex nuclear environment. In this proposal, several specific questions will be addressed concerning the structure and function of yeast nuclear RNase P. [1] How are RNase P substrates recognized by the RNA and protein components of this complex holoenzyme? [2] How are the subunits of the holoenzyme fit together to achieve this function? [3] What are the other roles of the protein subunits in physiological function of the holoenzyme within the nuclear tRNA processing pathway?