RNA BINDING SITE OF A TRANSLATIONAL REPRESSOR

描述：噬菌体MS2复制酶基因的翻译抑制 由于其外壳蛋白是研究最广泛和成果最多的系统之一 论蛋白质与RNA结合的特异性。蛋白质的结构 和RNA靶标是已知的，单独的和在复合体中。两个组件 它们都很小，可以大量制备，而且很容易结合 量化的。基因系统有助于蛋白质变体的选择 具有改变的专一性。最后，它是一个同源家族的成员 结合一组相似但显著不同的配体的蛋白质， 允许一种蛋白质的结构作为其他蛋白质的模型。 外壳蛋白-RNA复合体的晶体结构显示了两个碱基 MS2 RNA靶标，一个凸起的A在-10处和一个环A在-4处，结合在 准对称方式由四个或五个氨基酸残基组成 活性外壳蛋白二聚体的单体。这里提出的实验将 系统地探索每一种氨基酸的贡献 这些相互作用的链条，使用定点突变来 在这些位置用精心挑选的替代氨基酸来替代。《少年派》 已经表明，外壳蛋白二聚体可以被两个融合的单体取代 变成一个单一的多肽链。这允许对每个 相互独立的A-结合位点作为融合的杂二聚体。它有 稳定突变蛋白的进一步有用的特性是 以单体的形式稳定。 在以前的工作中，PI已经确定了RNA结合位点和外壳蛋白 相关噬菌体MS2、GA、Q-β和SP的特异性决定因素，以及 已经选择了变种，将一种蛋白质相互转化为 另一个核糖核酸。他现在提议将这些研究扩展到噬菌体PP7，这是 这个群体中关系最远的，没有数据的地方 表明外壳蛋白对复制酶有抑制作用。他将使用 与之前相同的两个质粒系统，其中外壳蛋白基因(靶向 用于突变)位于一个质粒和一个受噬菌体调控的lacZ基因上 另一种是翻译运算符。对转化子进行筛查，以确定其是否丢失 抑制和只有突变的蛋白质仍然能够组装成 选择衣壳类进行进一步研究。这会筛选出 导致错误折叠、降解或其他与RNA无关的变化 有约束力的。 最后，与以前的工作大不相同的是，国际和平研究所将启动一项 MS2包被物结合特异性的体外进化程序 蛋白。SELEX将被用来寻找最优的新RNA结构 用于结合特定的外壳蛋白变体。聚合型杂二聚体 构造将允许结合部位的两半不同 独立的。体外选择将扩展到蛋白质，通过 同时使用定点密码子随机化，DNA改组PCR， 和噬菌体展示。RNA目标最初将保持不变，并 通过部分互补生物素化的三明治固定化 奥戈。这些靶标被选为首选的结合位点 其他具有良好特性的RNA结合蛋白，具有某种变体的 在MS2衣壳中发现了β-折叠RNA结合域：U1a、HIV Tar和T4RegA。 这些RNA在结构上有明显的不同。最终， RNA和蛋白质成分的共同进化将被用来探索 这个蛋白质折叠中固有的RNA特异性的潜力。

DESCRIPTION: The translational repression of the phage MS2 replicase gene by its coat protein is one of the most widely studied and fruitful systems on the specificity of RNA binding by protein. The structures of the protein and RNA target are known, individually and in the complex. Both components are small, can be prepared in large quantity and their binding can be easily quantitated. A genetic system facilitates selection of protein variants with altered specificity. Finally, it is a member of a homologous family of proteins that bind a similar but significantly different set of ligands, allowing the structure of one protein to serve as the model for others. The crystal structure of the coat protein-RNA complex shows the two bases of the MS2 RNA target, a bulged A at -10 and a loop A at -4, are bound in a quasi symmetric way by four or five amino acid residues on each of the monomers of the active coat protein dimer. Experiments proposed here will systematically explore the contributions of each of the amino acid side chains to this these interactions, using site directed mutations to substitute carefully chosen alternate amino acids at these sites. The PI has shown that the coat protein dimer can be replaced by two monomers fused into a single polypeptide chain. This allows manipulation of each of the A-binding sites independently of the other as fused heterodimers. It has the further useful property of stabilizing mutant proteins that would not be stable as monomers. In previous work the PI has identified RNA binding sites and coat protein specificity determinants for the related phages MS2, GA, Q-beta and SP, and has selected variants that interconvert one protein to the specificity for another RNA. He now proposes to extend these studies to phage PP7, which is the most distantly related of this group and where there is no data to indicate that coat protein repression of replicase occurs. He will use the same two plasmid system as previously, where the coat protein gene (targeted for mutagenesis) is on one plasmid and a lacZ gene regulated by a phage translational operator on the other. Transformants are screened for loss of repression and only mutant proteins that are still able to assemble into capsids are chosen for further study. This screens out mutations that result in misfolding, degradation or other changes not relevant to RNA binding. Finally, in a major departure from previous work, the PI will initiate a program of in vitro evolution of the binding specificity of the MS2 coat protein. Selex will be used to find the new RNA structures that are optimal for binding to specific coat protein variants. The fused heterodimer construct will allow the two halves of the binding site to be varied independently. In vitro selection will be extended to the protein by the simultaneous use of site directed codon randomization, DNA shuffling PCR, and phage display. The RNA target will be held constant initially and immobilized through a sandwich with a partially complementary biotinylated oligo. These targets are chosen to be the preferred binding sites for other, well characterized RNA binding proteins that have some variant of the beta sheet RNA binding domain found in MS2 coat: U1A, HIV Tar and T4 RegA. These RNAs are significantly different in structure. Eventually, coevolution of both RNA and protein components will be used to explore the potential for RNA specificity inherent in this protein fold.