CONTROL OF CELL FUNCTION VIA SELECTIVE MRNA TRANSLATION

该项目的总体目标是帮助阐明 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的生物化学基础 （mRNA）翻译和分析特定的结构和功能， 核糖核蛋白复合物RNA-蛋白质复合物具有重要的 在许多生物过程中的酶和结构作用，包括 蛋白质生物合成、病毒复制和病毒组装。 模型 本项目中使用的系统是一组正义单链 RNA病毒、苜蓿花叶病毒和ILAR病毒，它们与 与导致严重脑炎的人类甲病毒有关。 的 病毒外壳蛋白mRNA翻译的调控及其特异性 和相对简单的病毒RNA外壳蛋白相互作用， 模型系统为研究提供了重要的优势。 三位有效 本供资期间的成绩是：（1）发现 病毒外壳蛋白中新的RNA结合共有序列，2） 通过使用化学修饰干扰， 和羟基自由基足迹分析， 病毒RNA上的位点，和3）3'非翻译的特征 作为病毒外壳蛋白mRNA翻译的关键调节因子的序列 效率更新提案的具体目标是定义 用于外壳蛋白-RNA相互作用的精确的RNA-蛋白接触， 阐明翻译控制的生化基础 由病毒mRNA 3'非翻译区介导。 一个假设， 可以测试的是，RNA结合共有序列中的一个关键精氨酸 RNA中的鸟嘌呤官能团。 将采用几种方法 绘制RNA-蛋白质接触图。 化学合成的RNA， 将分析特定碱基或核糖官能团修饰 在RNA结合实验中，包括天然凝胶电泳和 羟自由基足迹法 含氨基酸的肽 在高度保守位置的取代也将用于 确定结合的外壳蛋白是否具有α-螺旋特征或 芳香族氨基酸插入RNA螺旋中。 圆二色谱 数据表明外壳蛋白肽可以稳定RNA 结合时的构象变化。 一种灵敏的技术， 电双折射将用于表征构象 通过确定螺旋间角来改变。 一种遗传方法，基于 E.大肠杆菌，将用于快速筛选 功能获得性突变体以鉴定候选RNA-蛋白质接触。 我们认为3'非翻译区增强mRNA翻译， 通过募集翻译组分来竞争活性。 的 非洲爪蟾卵母细胞和酵母翻译系统被用于 图3' UTR序列和结构决定因素，使大衣 蛋白质mRNA竞争其他mRNA，这是病毒RNA使用的策略 夺取宿主的翻译器官 几种方法为高 提出了包括核磁共振和X射线衍射在内的结构解析方法 表达表面RNA的嵌合病毒颗粒的晶体学- 结合肽。

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.