Regulation of Eukaryotic Protein Synthesis Initiation

描述(由申请人提供)：本项目的长期目标是 了解在招募真核基因的过程中发生的步骤 核糖体及其调控方式。此过程决定了 蛋白质合成的总速度和翻译的mRNAs谱。 这四个具体目标主要涉及两个相互作用的启蒙因素 在这一过程中起核心作用的是eIF4E，它是一种结合 含7-甲基鸟苷的信使核糖核酸，以及连接eIF4G的蛋白质 所有已知参与信使核糖核酸募集的启动因子共同作用。这个 线虫为研究这种线虫提供了无与伦比的优势 作为蛋白质合成的起始的复杂的生化过程。令人惊讶的是， 它表达五种eIF4E亚型，称为IFE蛋白，具有不同的 帽子绑定属性。在具体目标1中，我们将尝试推断他们的 通过研究它们与m7G-和m3的生化作用来研究它们的生物学作用 含‘2’7G的帽类似物和寡核苷酸，与mRNA和其他 蛋白质，在后一种情况下利用MALDI-TOF质谱仪。具体而言 目的2，我们将确定人eIF4E磷酸化对蛋白质的影响 合成速率。EIF4G与eIF4E和Poly(A)结合蛋白结合， 两个C是作用于mRNA募集的效应子，然而eIF4G和eIF4G的许多亚型 在哺乳动物细胞中可观察到eIF4G基因的表达。在具体目标3中，我们将 确定这些eIF4G异构体的结构、功能和来源 描述已知的启动因子与它们的结合。以特定的目标 4、我们将确定eIF4G亚型在体内的功能，并具有特殊的 强调eIF4G裂解在小细胞瘤病毒诱导中的相对重要性 宿主关闭蛋白质合成。因为eIF4E和eIF4E的过度表达 EIF4G会导致细胞恶性转化，而且因为自然发生 肿瘤中含有大量升高的eIF4E，这些研究具有相关性 致癌。它们也与小核糖核酸病毒引起的疾病有关，如 如脊髓灰质炎病毒(脊髓灰质炎)、鼻病毒(普通感冒)和柯萨奇病毒(心脏 失败)。

DESCRIPTION (provided by applicant): The long-term goals of this project are to understand the steps that occur during the recruitment of eukaryotic mRNA to the ribosome and how they are regulated. This process determines both the overall rate of protein synthesis and also the spectrum of mRNAs translated. The four Specific Aims principally concern two interacting initiation factors that are central to this process: eIF4E, the protein that binds the 7-methylguanosine-containing cap of mRNA, and eIF4G, the protein that links together all initiation factors known to be involved in mRNA recruitment. The nematode C. elegans provides unparalleled advantages for studying such a complex biochemical process as initiation of protein synthesis. Surprisingly, it expresses five eIF4E isoforms, termed IFE proteins, which have differing cap-binding properties. In Specific Aim 1, we wi2ll attempt to infer their biological roles by studying their biochemical interactions with m7G- and m3 '2'7G-containing cap analogues and oligonucleotides, with mRNAs, and with other proteins, utilizing MALDI-TOF mass spectrometry in the latter case. In Specific Aim 2, we will determine the effect of human eIF4E phosphorylation on protein synthesis rate. eIF4G binds to both eIF4E and the poly(A)-binding protein, the two C is-acting effectors for mRNA recruitment, yet many isoforms of eIF4G and eIF4G mRNA are observed in mammalian cells. In Specific Aim 3, we will determine the structure, function, and origin of these eIF4G isoforms and characterize the binding of known initiation factors to them. In Specific Aim 4, we will determine the function of eIF4G isoforms in vivo, with special emphasis on the relative importance of eIF4G cleavage in picomavirus-induced host shut-off of protein synthesis. Because overexpression of both eIF4E and eIF4G causes malignant transformation of cells, and because naturally occurring tumors contain greatly elevated levels of eIF4E, these studies have relevance to cancer. They also have relevance for diseases caused by picornaviruses such as poliovirus (polio), rhinovirus (the common cold) and Coxsackievirus (heart failure).