Structure and Function of the Decapping Enzyme Complex

项目摘要摘要 信使核糖核酸的破坏是真核基因表达中的关键事件，在早期动物中起着至关重要的作用 发育、细胞生长、增殖和对压力的适应。专用的mRNA稳定途径 确保包含提前终止密码子的异常转录被消除，并确保密钥的丰富 转录本，如那些编码细胞因子、白介素类和原癌基因的转录本，受到严格控制。一个 这些途径中的关键调控步骤是由Dcp2去除5‘末端帽结构，这是 通过将信使核糖核酸小体的5‘一磷酸暴露于5’至3‘来判定信使信使被破坏 外切核糖核酸酶。Dcp2的活性由多种途径特异性的共激活剂通过 机制还不是很清楚。以发芽酵母为模型系统，我们将结合 生化、生物物理和遗传学方法确定必需激活物Dcp1如何调节 Dcp2的催化活性。在特定目标1中，Dcp1/Dcp2与非 可水解底物将指导突变株的动力学和遗传分析，以剖析 在特定的目标2中，我们将使用核磁共振波谱来确定Dcp1是否增强了催化 通过在非活性形式和活性形式之间改变构象平衡来激活Dcp2的活性；在特定目标3中， 我们将确定Dcp1/Dcp2与增强剂Dcp1/Dcp2三元配合物的晶体结构 解帽蛋白家族成员Edc1，以确定共激活剂刺激的机制。这些 综合研究将揭示几个影响mRNA稳定途径的关键步骤 数以千计的人类基因。

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.