Structure and Function of the Decapping Enzyme Complex

脱帽酶复合物的结构和功能

• 批准号: 8387778
• 负责人: John D Gross
• 金额: $ 24.87万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8387778
• 关键词: Address; Affect; Animals; Binding; Biochemical; Biochemical Genetics; Biological Assay; Biological Models; Carbon; Catalysis; Chemicals; Chemistry; Code; Commit; Complex; Data; Development; Dinucleoside Phosphates; Enhancers; Ensure; Equilibrium; Event; Excision; Family member; Fission Yeast; Gene Expression; Genes; Genetic; Goals; Guanosine; Hereditary Disease; Human; Hydrolysis; In Vitro; Inherited; Interleukins; Kinetics; Laboratories; Lesion; Light; Link; Malignant Neoplasms; Measures; Mental Retardation; Messenger RNA; Metabolism; Metals; Methods; Molecular; Molecular Conformation; Multienzyme Complexes; Mutation; N-terminal; NMR Spectroscopy; Nonsense Codon; Nucleotides; Pathway interactions; Play; Process; Protein Family; Proteins; Proto-Oncogenes; RNA; RNA Binding; RNA Cap-Binding Proteins; RNA Decay; RNA Splicing; RNA Stability; Reaction; Regulation; Resolution; Role; Saccharomycetales; Source; Stress; Structure; Substrate Specificity; Testing; Transcript; Translations; Yeasts; abstracting; analog; base; cancer genetics; cell growth; conformational conversion; cytokine; decapping enzyme; genetic analysis; human disease; mutant; phosphodiester; poly A specific exoribonuclease; research study; yeast genetics

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.

项目摘要摘要 mRNA的破坏是真核基因表达的关键事件，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mRNA稳定性途径 确保消除包含过早停止密码子的异常笔录和大量的密钥 诸如编码细胞因子，白细胞介绍和原始基因的转录本受到严格控制。一个 在这些途径中，关键，调节的步骤是通过DCP2去除5'端子盖结构， 句子通过将mRNA体的5'单磷酸盐暴露于5'至3'来破坏mRNA 驱虫。 DCP2的活性通过多种途径特异性共激活因子刺激 不太了解的机制。使用芽酵母作为模型系统，我们将结合 生化，生物物理和遗传学方法来确定必需激活剂DCP1如何调节 DCP2的催化活性。在特定的目标1中，DCP1/DCP2复合物的晶体学研究与非 - 可水解底物将指导突变体的动力学和遗传分析，以剖析化学步骤 dec在特定的目标2中，我们将使用NMR光谱来确定DCP1是否增强了催化 DCP2的活性通过在非活性和活性形式之间移动构象平衡；在特定的目标3中 我们将确定DCP1/DCP2之间三元络合物的晶体结构 dectaping蛋白家族成员EDC1，以定义共激活因子刺激的机理。这些 综合研究将阐明几个影响影响途径的mRNA稳定途径的关键步骤 成千上万的人类基因丰富。