Biophysical Studies of RNase P Protein Folding

描述(申请人提供)：核糖核蛋白，或RNA/蛋白质 复合体(RNPs)与多种人类疾病有关，包括 自身免疫性疾病，如狼疮和关节炎。RNPs是必不可少的组成部分 在所有的细胞中。它们在许多细胞功能中发挥着关键作用，但也许 最重要的是蛋白质的合成。RNPs参与转录； 也就是说，它们是核糖体的主要组成部分，在那里蛋白质翻译 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)，它去除了未成熟的RNA分子的5‘前导，以 产生成熟的tRNA。细菌核糖核酸酶P由一个300-400个核苷酸RNA组成 亚基和1-120残基蛋白分子。人们对此知之甚少 RNPs组装的一般细胞机制和RNaseP 具体地说。拟议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，蛋白质亚单位是 在没有阴离子配体的情况下变性。Oas博士假设RNase 为了增强P蛋白的能力，P蛋白已经进化到天然地展开。 组装成全酶复合体，或通过动力学或通过增加 其与RNaseP RNA相互作用的特异性。OAS博士将对此进行测试 通过比较小分子和寡核苷酸结合的假说。OAS博士 也将决定蛋白质三种状态的相对能量： 变性的、未连接的原生体和连接的原生体。OAS博士还将测量 折叠和结合的相对速度，并验证他的假设 结合发生在折叠之后。此外，他还将比较 将天然蛋白质去连接到与小分子配体结合的蛋白质， 寡核苷酸和核糖核酸酶P-RNA。这项体外工作将使未来成为可能 RNaseP和其他RNPs在细胞内组装的实验。

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.