Structure and Function of the Decapping Enzyme Complex

项目摘要 mRNA的破坏是真核生物基因表达的关键事件，在早期动物中起着重要作用 发育、细胞生长、增殖和对压力的适应。专用mRNA稳定性途径 确保含有提前终止密码子的异常转录物被消除， 转录物如编码细胞因子、白细胞介素和原癌基因的转录物受到严格控制。一 这些途径中的关键调节步骤是通过Dcp 2去除5'端帽结构， 通过将mRNA体的5 ′单磷酸暴露于5 ′至3 ′磷酸， 核糖核酸外切酶Dcp 2的活性通过多种途径特异性共激活剂刺激， 这些机制还没有被很好地理解。以芽殖酵母为模型系统，我们将联合收割机 生物化学、生物物理学和遗传学方法来确定必需激活剂Dcp 1如何调节 Dcp 2的催化活性。在具体目标1中，Dcp 1/Dcp 2复合物与非- 可水解底物将指导突变体的动力学和遗传分析，以剖析 在具体的目标2中，我们将使用NMR光谱来确定Dcp 1是否增强催化脱帽; 通过在非活性和活性形式之间改变构象平衡来提高Dcp 2的活性;在具体目标3中， 我们将确定Dcp 1/Dcp 2与增强剂之间的三元复合物的晶体结构， 脱帽蛋白家族成员Edc 1，以确定共激活剂的刺激机制。这些 综合研究将揭示几种mRNA稳定性途径中的关键步骤， 丰富的人类基因。

Project Summary Abstract The destruction of mRNA is a key event in eukaryotic gene expression, playing a crucial role in early animal development, cellular growth , proliferation and adaptation to stress. Dedicated mRNA stability pathways ensure that aberrant transcripts containing premature stop codons are eliminated and the abundance of key transcripts such as those coding for cytokines, interleukins and proto-oncogenes are tightly controlled. A critical, regulated step in these pathways is the removal of the 5' terminal cap structure by Dcp2, which sentences an mRNA for destruction by exposing the 5' monophosphate of the mRNA body to 5' to 3' exoribonucleases. The activity of Dcp2 is stimulated by a variety of pathway specific co-activators through mechanisms that are not well understood. Using budding yeast as a model system, we will combine biochemical , biophysical and genetic methods to determine how the essential activator Dcp1 regulates the catalytic activity of Dcp2. In specific aim 1, crystallographic studies of the Dcp1/Dcp2 complex with non- hydrolyzable substrate will guide kinetic and genetic analyses of mutants to dissect the chemical step of decapping ; in specific aim 2, we will use NMR spectroscopy to determine if Dcp1 enhances the catalytic activity of Dcp2 by shifting a conformational equilibrium between inactive and active forms; in specific aim 3, we will determine the crystal structure of the ternary complex between Dcp1/Dcp2 with the Enhancer of decapping protein family member , Edc1, to define the mechanism of stimulation by co-activators. These integrated studies will shed light on a critical step in several mRNA stability pathways that affects the abundance of thousands of human genes.