CONTROL OF CELL FUNCTION VIA SELECTIVE MRNA TRANSLATION

这个项目的广泛目标是有助于阐明 人类疾病的遗传基础。这项工作的近期目标是 确定选择性和竞争性信使RNA的生化基础 (信使核糖核酸)的翻译，并分析特定的结构和功能 核糖核蛋白复合体。RNA-蛋白质复合体具有必不可少的 酶和结构在许多生物过程中的作用，包括 蛋白质生物合成、病毒复制和病毒组装。模型 本项目使用的系统是一组正向单链 RNA病毒、紫花苜蓿花叶病毒和丁香花叶病毒，它们关系密切 与导致严重脑炎的人类甲型病毒有关。这个 病毒外壳蛋白mRNA翻译的调控及其特异性 以及病毒RNA-外壳蛋白相互作用的相对简单 模型系统为研究提供了重要的优势。三大意义 本供资期间取得的成就是：1)发现 病毒外壳蛋白中一种新的RNA结合共识序列，2) 通过使用化学修饰干扰进行识别 和羟基自由基足迹分析，对外壳蛋白的结合 3)3‘非编码区的特性 序列作为病毒外壳蛋白mRNA翻译的关键调节因子 效率。续签提案的具体目标是界定 外壳蛋白-RNA相互作用的精确RNA-蛋白质接触 阐明翻译调控的生化基础 由病毒mRNA3‘非翻译区介导。一个假设是 被测试的一个关键是精氨酸在RNA中结合的共识接触 RNA中的鸟嘌呤官能团。将使用几种方法 来绘制RNA-蛋白质接触图。化学合成的RNA，含有 将分析特定的碱基或核糖官能团修饰 在RNA结合实验中，包括天然凝胶电泳法和 羟基自由基足迹。含氨基酸的多肽 在高度保守的位置上的替换也将用于 确定结合的外壳蛋白是否具有α-螺旋特性 芳香氨基酸嵌入到RNA螺旋中。圆二向色性 数据表明，外壳蛋白多肽可以稳定rna。 结合时的构象变化。一种敏感的技术，瞬变的 电双折射，将被用来表征构象 通过确定螺旋角来改变。一种遗传方法，基于 在大肠杆菌中的翻译抑制，将被用于快速筛选 功能获得突变体，以确定候选的RNA-蛋白质接触。 我们认为，3‘端非翻译区增强了信使核糖核酸的翻译， 通过招聘翻译组件开展竞争活动。这个 非洲爪哇的卵母细胞和酵母翻译系统正被用于 MAP 3‘非编码区序列和结构决定因素使涂层 蛋白质信使核糖核酸胜过其他信使核糖核酸，这是病毒核糖核酸使用的策略 篡夺东道主的翻译设备。实现高效率的几种方法 提出了包括核磁共振和X-射线在内的分辨结构分析 表达表面RNA的嵌合病毒颗粒的结晶学 结合多肽。

The broad goal of this project is to contribute to elucidating the genetic basis of human disease. The immediate goals of the work are to define the biochemical basis of selective and competitive messenger RNA (mRNA) translation and to analyze the structure and function of specific ribonucleoprotein complexes. RNA-protein complexes have essential enzymatic and structural roles in many biological processes, including protein biosynthesis, virus replication, and virus assembly. The model system used in this project is a group of positive-sense single-stranded RNA viruses, alfalfa mosaic virus and ilarviruses, which are closely related to human alphaviruses that cause severe encephalitis. The regulation of viral coat protein mRNA translation and the specificity and relative simplicity of viral RNA-coat protein interactions in this model system offer important advantages for study. Three significant accomplishments of the current funding period are 1) the discovery of a novel RNA binding consensus sequence in the viral coat proteins, 2) the identification, through use of chemical modification interference and hydroxyl radical footprint analysis, of the coat protein binding site on viral RNA, and 3) the characterization of 3' untranslated sequences as key regulators of viral coat protein mRNA translational efficiency. The Specific Aims of the renewal proposal are to define precise RNA-protein contacts for the coat protein-RNA interaction and to elucidate the biochemical basis for the translational control mediated by the viral mRNA 3' untranslated region. One hypothesis to be tested is that a key arginine in the RNA binding consensus contacts guanine functional groups in the RNA. Several approaches will be used to map RNA-protein contacts. Chemically synthesized RNAs containing specific base or ribose functional group modifications will be analyzed in RNA binding experiments, including native gel electrophoresis and hydroxyl radical footprinting. Peptides containing amino acid substitutions at highly conserved positions will also be used in determining if bound coat protein has alpha-helical character or if aromatic amino acids intercalate in the RNA helices. Circular dichroism data suggest that a coat protein peptide may stabilize an RNA conformational change upon binding. A sensitive technique, transient electric birefringence, will be used to characterize the conformational change by determining interhelical angles. A genetic method, based on translational repression in E. coli, will be used to rapidly screen gain-of-function mutants to identify candidate RNA-protein contacts. We propose that the 3' untranslated region enhances mRNA translation and competitive activity by recruiting translational components. The Xenopus laevis oocyte and yeast translation systems are being used to map 3' UTR sequence and structural determinants that enable the coat protein mRNA to out-compete other mRNAs, a strategy used by viral RNAs to usurp the host translational apparatus. Several approaches for high resolution structural analysis are proposed, including NMR and X-ray crystallography of chimeric virus particles expressing surface RNA- binding peptides.