Functions of mRNP Proteins in the Process of Translation

mRNA翻译调控是调控哺乳动物细胞基因表达的一个重要方面。翻译控制的大多数例子涉及蛋白质合成起始阶段的调节。可以区分两种起始事件，其中任何一种都可以调节：1)在多聚体上起始，从而维持mrna上核糖体的完整补充；2)在信使核糖核蛋白（mRNP）颗粒上起始，从而将非活性mrna动员到活性多体中。人们对后一种情况知之甚少，但对它的管制却经常遇到。pi建议表征与mRNP颗粒结合并掩盖其活性的蛋白质。在普什奇诺中已纯化出一个50 kDa的蛋白，并将对其作为翻译抑制剂的作用机制进行探讨。将确定其与mrna的结合位点，并确定蛋白质合成的抑制步骤。他们还计划克隆其cDNA，并确定在转染的哺乳动物细胞中过度表达的影响。来自Puschino的初步证据表明，50 kDa蛋白本身可能受到一种“激活剂”的调节；将对“活化剂”进行纯化和表征。这些研究依赖于Davis和Puschino目前使用的蛋白质纯化和化学的经典生化技术，采用体外无细胞翻译系统，使用Davis纯化的起始因子进行高度分离和重组，并采用最先进的转染方法。该项目旨在阐明mRNP颗粒如何被激活和抑制翻译的基本分子机制，从而有助于阐明这一主要的鲜为人知的基因调控领域。

Regulation of mRNA translation constitutes an important aspect in the control of gene expression mammalian cells. Most examples of translational control involve modulation of the initiation phase of protein synthesis. Two kinds of initiation events can be distinguished either of which may be regulated: 1) initiation on polysomes, thereby maintaining a full complement of ribosomes on the mRNAs; and 2) initiation on messenger ribonucleoprotein (mRNP) particles, thereby mobilizing non- active mRNAs into active polysomes. The latter event is poorly understand, yet its regulation is encountered frequently. The PIs propose to characterize proteins that bind to mRNP particles and that mask their activiies. One such protein of 50 kDa has been purified in Pushchino, and its mechanism of action as a translational inhibitor will be probed. Its site of binding to mRNAs will be determined, and the inhibited step of protein synthesis will be identified. They also plan to clone its cDNA and determine the effect of overexpression in transfected mammalian cells. Preliminary evidence from Puschino suggests that the 50 kDa protein may itself be regulated by an "activator"; the "activator" will be purified and characterized. These studies rely on classical biochemical techniques of protein purification and chemistry presently used in both Davis and Puschino, employ in vitro cell-free translatiion systems, some highly fractionated and reconstituted with purified initiatiion factors from Davis, and employ state-of-the-art transfection methods. %%% The project is designed to shed light on the basic molecular mechanisms of how mRNP particles are activated and inhibited for translation, and thus should help elucidate this major poorly understood area of gene regulation.