CONTROL OF CELL FUNCTION VIA SELECTIVE MRNA TRANSLATION

要完全了解疾病的分子机制，需要 阐明基因调控机制。这份提案的重点是 翻译水平的基因控制机制和实验是直接的 核糖核酸的生物化学和结构/功能关系 酸(RNA)和特定的RNA-蛋白质相互作用。这样做的目的是 项目是定义选择性信使RNA的分子基础 (信使核糖核酸)翻译，并表征存在于RNA和 形成特定核糖核蛋白复合体的蛋白质。 研究的模式系统以紫花苜蓿花叶病毒(AlMV)为中心， 其基因排列和蛋白质序列与 会导致严重脑炎的人类甲型病毒。AlMV RNA4，病毒 外壳蛋白信使RNA，是一个高效翻译的模板，并且 翻译的外壳蛋白与3‘非翻译区特异性结合 它的信使RNA。这种RNA-蛋白质相互作用是两种物质所必需的 病毒组装和病毒复制；此外，迄今为止的进展 这表明它对它的高效翻译也可能是重要的。删除 AlMV RNA4的3‘非翻译区，包括外壳 蛋白结合域，严重影响mRNA翻译 效率。对照实验表明，减少平移并不是 由于mRNAs的不稳定。一种39个核苷酸的最小外壳蛋白 已在AlMV RNA4的3‘端确定了结合位点，它 已经证明，外壳蛋白的氨基末端既有 与RNA结合的必要条件和充分条件。的功能意义 通过显示涂层来确认氨基末端RNA结合域 蛋白质多肽(长度为25或38个氨基酸)与RNA特异性结合， 改变RNA构象，激活病毒的初始步骤 复制。多肽结合时观察到的RNA构象变化 与观察到的构象变化非常相似 人类免疫缺陷病毒(HIV)的Tat和Rev蛋白与其RNA结合 目标。续展期的具体目标包括 AlMV RNA43‘非翻译区在人类基因组中的作用 协助翻译。最小的3‘序列或结构将 保持高效的翻译将通过删除分析来映射； 此外，外壳蛋白结合对RNA翻译的影响 效率将受到考验。这些实验与努力很好地结合在一起。 针对AlMV结合多肽的生化和生物物理分析 RNA4片段。随机多肽文库与体外实验的结合 RNA配体的选择将被用来精确定义RNA和蛋白质 决定因素，以及RNA和RNA-蛋白质复合体的结构 被化学干扰研究和羟基自由基探查 脚印。用葡萄藤对配合物进行了功能分析 植物原生质体中的复制分析。生化研究是 足够先进，我们已经启动了对 多肽-RNA复合体的核磁共振谱，并试图通过培养共 与RNA结合的多肽晶体。

A complete understanding of the molecular mechanisms of disease requires elucidation of gene regulatory mechanisms. This proposal focuses upon translation-level gene control mechanisms and the experiments are directed at the biochemistry and structure/function relationships of ribonucleic acids (RNA) and specific RNA-protein interactions. The goals of this project are to define the molecular basis of selective messenger RNA (mRNA) translation and to characterize determinants present in RNAs and proteins that underlie formation of specific ribonucleoprotein complexes. The model system for study centers upon alfalfa mosaic virus (AlMV), the gene arrangement and protein sequences of which show similarities to the human alphaviruses that cause severe encephalitis. AlMV RNA 4, the viral coat protein messenger RNA, is an efficiently-translated template, and the translated coat protein binds specifically to the 3' untranslated region of its messenger RNA. This RNA-protein interaction is required both for virus assembly and for virus replication; moreover, progress to date suggests it may also be important for its efficient translation. Deletion of the 3' untranslated region of AlMV RNA 4, which includes the coat protein binding domain, severely compromises mRNA translational efficiency. Control experiments show that diminished translation is not due to destabilization of the mRNAs. A 39-nucleotide minimal coat protein binding site has been identified at the 3' terminus of AlMV RNA 4, and it has been demonstrated that the amino-terminus of the coat protein is both necessary and sufficient for binding RNA. The functional significance of the amino-terminal RNA binding domain was confirmed by showing that coat protein peptides (25 or 38 amino acids in length) bind RNA specifically, alter RNA conformation, and activate the initial steps of virus replication. The changes in RNA conformation observed upon peptide binding are very similar to conformational changes observed when peptides from the Tat and Rev proteins of human immunodeficiency virus (HIV) bind their RNA targets. The specific aims for the continuation period include characterizing the role of the AlMV RNA 43' untranslated region in facilitating translation. The minimal 3' sequence or structure that will maintain efficient translation will be mapped by deletion analysis; moreover, the effect of coat protein binding on RNA translational efficiency will be tested. These experiments couple well with efforts aimed at a biochemical and biophysical analysis of peptide binding to AlMV RNA 4 fragments. A combination of random peptide libraries and in vitro selection of RNA ligands will be used to precisely define RNA and protein determinants, and the structure of the RNA and RNA-protein complexes will be probed by chemical interference studies and hydroxyl radical footprinting. Functional analysis of the complexes is tested by vines replication assays in plant protoplasts. The biochemical studies are sufficiently advanced that we have initiated structural analysis of the peptide-RNA complex by NMR spectroscopy and by attempting to grow co- crystals of peptide bound to RNA.