RNA-BINDING SITE OF A TRANSLATIONAL REPRESSOR

越来越多的人认识到，特异性RNA的识别 蛋白质序列是一个基本方面的各种正常的 细胞过程 更多的注意力集中在这一点上 由于RNA-蛋白质相互作用的关键作用， 人类免疫缺陷病毒的生命周期，艾滋病的原因。 的 这项工作的长期目标是促进对 RNA-蛋白质相互作用的基本性质， 这种互动的具体模式。 的外壳蛋白 细菌病毒MS 2是一种遗传调节蛋白。 它绑定了一个 特异性RNA茎环（翻译操纵子）抑制翻译 病毒复制酶基因。 本实验室以前的工作已经定义了 通过分离外壳蛋白突变体的RNA结合位点， 对RNA的亲和力降低，对RNA的亲和力增加，或 显示改变的RNA结合特异性。 目前的主要目标 我们的建议是将我们对这些结构的不同知识联合收割机结合起来， 外壳蛋白的RNA结合位点及其RNA配体如何结合到一个图片中， 两个分子相互作用。 提出了两种一般方法。 第一、 因为突变蛋白必须通过去除 联系人或添加新的联系人，这些丢失或添加的联系人的具体性质 可以通过询问突变蛋白质与野生型蛋白质的不同来探测接触 在它们适应化学修饰或核苷酸的能力方面 在RNA中的特定位点进行取代。 第二，进化变异 的MS 2病毒利用同源外壳蛋白结合不同的RNA 结构. 建议定义蛋白质结构决定簇 RNA结合特异性的基因和生物化学的结合， 方法.

It is increasingly appreciated that the recognition of specific RNA sequences by proteins is a fundamental aspect of a variety of normal cellular processes. Additional attention has been focussed on this problem because of the critical roles played by RNA-protein interactions in the life cycle of Human Immunodeficiency Virus, the cause of AIDS. The long term goal of this work is to contribute to a general understanding of the fundamental nature of RNA-protein interactions by understanding one particular model of such an interaction in detail. The coat protein of the bacterial virus MS2 is a genetic regulatory protein. It binds a specific RNA stem-loop (the translational operator) to repress translation of the viral replicase gene. Previous work in this laboratory has defined the RNA binding site through the isolation of coat protein mutants which either have reduced affinity for RNA, have increased affinity for RNA, or show altered RNA binding specificity. The main objective of the present proposal is to combine our separate knowledge of the structures of the coat protein RNA-binding site and its RNA ligand into a picture of how the two molecules interact. Two general approaches are proposed. First, since the mutant proteins must exert their effects by either removing contacts or adding new ones, the specific nature of these missing or added contacts can be probed by asking how mutant proteins differ from wild-type in their abilities to accommodate chemical modification or nucleotide substitution at specific sites in the RNA. Second, evolutionary variants of the MS2 virus utilize homologous coat proteins to bind divergent RNA structures. It is proposed to define the protein structural determinants of RNA binding specificity using a combination of genetic and biochemical methods.