RNA BINDING SITE OF A TRANSLATIONAL REPRESSOR

描述：噬菌体MS 2复制酶基因的翻译抑制 它的外壳蛋白是一个最广泛研究和富有成效的系统 RNA与蛋白质结合的特异性。 蛋白质的结构 和RNA靶标是已知的，单独地和在复合物中。 两种组分 小，可以大量制备，并且它们的结合可以容易地进行， 定量。 遗传系统有助于选择蛋白质变体 改变了特异性。 最后，它是一个同源家族的成员， 结合相似但显著不同的配体组的蛋白质， 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复合物的晶体结构显示了 MS 2 RNA靶，在-10处凸出A和在-4处的环A，以 在每一个上通过四个或五个氨基酸残基的准对称方式， 活性外壳蛋白二聚体的单体。 这里提出的实验将 系统地探索每个氨基酸侧的贡献 链到这些相互作用，使用定点突变， 在这些位点替换精心选择的替代氨基酸。 的PI 已经表明外壳蛋白二聚体可以被两个融合的单体所取代， 变成一条多肽链 这允许操纵每个 A-结合位点独立于其他位点作为融合的异源二聚体。 它有 稳定突变蛋白质的另一个有用的特性是， 作为单体稳定。 在以前的工作中，PI已经确定了RNA结合位点和外壳蛋白 - 相关的DMS 2、GA、Q-β和SP的特异性决定因素，以及 已经选择了将一种蛋白质相互转化为特异性的变体， 另一个RNA 他现在建议将这些研究扩展到噬菌体PP 7， 这群人中最远的亲戚，没有数据 表明发生了复制酶外壳蛋白阻遏。 他将使用 与先前相同的双质粒系统，其中外壳蛋白基因（靶向 用于诱变）在一个质粒上，并且lacZ基因由噬菌体调节 translational operator另一方面 筛选转化体的缺失。 只有突变蛋白仍然能够组装成 选择衣壳用于进一步研究。 这会筛选出 导致错误折叠、降解或其他与RNA无关的变化 约束力 最后，与以前的工作有很大不同，PI将启动一个 MS 2包被结合特异性的体外进化程序 蛋白 Selex将用于寻找最佳的新RNA结构 用于与特异性外壳蛋白变体结合。 融合的异二聚体 构建体将允许结合位点的两个半部分变化 独立地。 体外选择将通过以下方法扩展到蛋白质： 同时使用定点密码子随机化，DNA改组PCR， 和噬菌体展示。 RNA靶标最初将保持恒定， 通过与部分互补的生物素化的 寡。 选择这些靶标作为优选的结合位点， 其他，充分表征的RNA结合蛋白，具有一些变体的RNA结合蛋白， 在MS 2外壳中发现的β折叠RNA结合域：U1 A、HIV Tar和T4 RegA。 这些RNA在结构上明显不同。 最后， RNA和蛋白质组分的共同进化将用于探索 这种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.