Biophysical Studies of RNase P Protein Folding

描述（由申请人提供）：核糖核蛋白或RNA/蛋白 核糖核酸复合物（RNP）与几种人类疾病有关，包括 自身免疫性疾病，如狼疮和关节炎。RNP是必不可少的组件 所有的细胞。它们在许多细胞功能中起着关键作用，但也许 最重要的是蛋白质合成。RNP参与转录; 也就是说，它们是核糖体的主要组成部分， 发生，它们负责产生成熟的转移RNA（tRNA） 分子。这最后一个基本功能是由核糖核酸酶执行的 P（RNase P），其去除未成熟RNA分子的5'前导序列， 产生成熟的tRNA细菌核糖核酸酶P由一个300-400核苷酸的RNA组成， 亚基和一个-120残基蛋白分子。关于这一点，我们知之甚少。 一般RNP和RNaseP组装的细胞机制 具体来说所提出的研究的目标是阐明动力学和 生理条件下细菌RNaseP组装的热力学机制 条件Oas博士的初步研究表明， 在不存在阴离子配体的情况下变性。Oas博士假设RNase P蛋白已经进化成天然未折叠的，以增强其能力， 组装成全酶复合物，无论是动力学上还是通过增加 它与RNaseP RNA相互作用的特异性。奥斯博士会测试这个 假设通过比较小分子和寡核苷酸结合。奥斯博士 也将决定蛋白质三种状态的相对能量： 变性的、未配体的天然的和配体的天然的。Oas博士还将测量 折叠和结合的相对速率，并测试他的假设， 在折叠之后发生结合。此外，他还将比较 未配体的天然蛋白质与与小分子配体结合的蛋白质相比， 寡核苷酸和RNase P RNA。这项体外研究将使未来 RNaseP和其他RNP在细胞中组装的实验。

DESCRIPTION (provided by applicant): Ribonucleoproteins, or RNA/protein complexes (RNPs), are associated with several human diseases including autoimmune disorders such as lupus and arthritis. RNPs are essential components of all cells. They play a critical role in many cellular functions, but perhaps the most important is in protein synthesis. RNPs are involved in transcription; i.e., they are the primary component of the ribosome, where protein translation occurs, and they are responsible for producing of mature transfer RNA (tRNA) molecules. This last essential function is performed by the enzyme ribonuclease P (RNase P), which removes the 5' leader of the premature RNA molecule to produce a mature tRNA. Bacterial RNase P consists of one 300-400 nucleotide RNA subunit and one -120 residue protein molecule. Very little is known about the cellular mechanisms for the assembly of RNPs in general and RNaseP specifically. The goal of the proposed research is to elucidate the kinetic and thermodynamic mechanisms of bacterial RNaseP assembly under physiological conditions. Dr. Oas' preliminary studies have shown that the protein subunit is denatured in the absence of an anionic ligand. Dr. Oas hypothesizes that RNase P protein has evolved to be natively unfolded, in order to enhance its ability to assemble into the holoenzyme complex, either kinetically or by increasing the specificity of its interaction with RNaseP RNA. Dr. Oas will test this hypothesis by comparing small molecule and oligonucleotide binding. Dr. Oas will also determine the relative energies of three states of the protein: denatured, unliganded native and liganded native. Dr. Oas will also measure the relative rates of folding and binding and test his hypothesis that ligand binding occurs after folding. In addition, he will compare the structures of unliganded native protein to that of protein bound to small molecule ligands, olgonucleotides and RNase P RNA. This in vitro work will make possible future experiments on the assembly of RNaseP and other RNPs in the cell.