CONTROL OF CELL FUNCTION VIA SELECTIVE MRNA TRANSLATION

要完全了解疾病的分子机制， 阐明基因调控机制。该提案的重点是 基因水平的调控机制和实验指导 核糖核酸的生物化学和结构/功能关系 核酸（RNA）和特定的RNA-蛋白质相互作用。这个的目标 该项目的目的是确定选择性信使RNA的分子基础， （mRNA）翻译和表征RNA中存在的决定簇， 构成特定核糖核蛋白复合物基础的蛋白质类。 研究的模型系统以苜蓿花叶病毒（AlMV）、苜蓿花叶病毒（AlMV）和苜蓿花叶病毒（AlMV）为中心。 其基因排列和蛋白质序列显示出与 导致严重脑炎的人类甲病毒。AlMV RNA 4，病毒 外壳蛋白信使RNA是一种有效的翻译模板， 翻译的外壳蛋白特异性结合3'非翻译区 它的信使RNA。这种RNA-蛋白质相互作用是必需的， 病毒组装和病毒复制;此外，迄今为止的进展 这也可能对它的高效翻译很重要。删除 AlMV RNA 4的3'非翻译区，包括外壳 蛋白结合域，严重损害mRNA翻译 效率对照实验表明，减少翻译不是 这是由于mRNA的不稳定。一种39个核苷酸的最小外壳蛋白 在AlMV RNA 4的3'末端鉴定出一个结合位点， 已经证明，外壳蛋白的氨基末端是 这是结合RNA的必要条件和充分条件。的功能意义 氨基末端RNA结合结构域通过显示包被 蛋白质肽（长度为25或38个氨基酸）特异性结合RNA， 改变RNA构象，激活病毒的初始步骤， 复制的肽结合后观察到的RNA构象变化 非常类似于当肽来自 人类免疫缺陷病毒（HIV）的达特和Rev蛋白结合其RNA 目标的延续期的具体目标包括 表征AlMV RNA 43'非翻译区在以下中的作用： 方便翻译。最小的3'序列或结构， 保持高效翻译将通过删除分析映射; 此外，外壳蛋白结合对RNA翻译的影响， 效率将受到考验。这些实验与努力相结合 旨在对与AlMV结合的肽进行生物化学和生物物理分析 RNA 4片段。随机肽库和体外肽库的组合 RNA配体的选择将用于精确定义RNA和蛋白质 决定簇，并且RNA和RNA-蛋白质复合物的结构将 通过化学干扰研究和羟基自由基 脚印通过藤蔓测试了复合物的功能分析 植物原生质体中的复制测定。生化研究是 足够先进，我们已经开始了结构分析， 肽-RNA复合物通过NMR光谱和试图生长共- 与RNA结合的肽晶体。

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.