Activities of yeast Ccr4-Not transcription factor complex

摘要： 我的研究项目的目标是了解多功能蛋白质复合物如何调节基因 表达，尤其是在应激反应中。我们目前的重点是酵母Ccr 4-不复杂， 几乎涉及基因控制的所有方面，包括转录，mRNA降解，翻译 阻遏和蛋白质泛素化。这个复合体在所有真核生物中都高度保守。本实验室 在理解其在转录中的作用，特别是RNA聚合酶II延伸方面， 采用多方面的方法，包括遗传学、分子生物学、重组生物化学和 基因组学描述Ccr 4-Not需要一个灵活的策略和向新方向发展的意愿。 这项提议将解决两个不同的过程在其控制下，即其泛素化活动的作用 基因表达和应激过程中Ccr 4-Not mRNA靶点重编程的机制 作为平衡mRNA合成和衰变的手段。 转录和DNA损伤反应过程中的蛋白质破坏是一个重要的过程。的not 4 复合物的亚基包含E3 RING结构域，并控制蛋白质的泛素依赖性破坏。 我们已经表明，Ccr 4-Not与延伸复合物相关，最近，它调节了细胞的生长， DNA损伤后RNAPII的破坏。主题1将使用全局蛋白质组学识别Not 4的新靶点， 确定修改的位置，并使用分子遗传学和生物化学来确定结果 对蛋白质功能的影响。识别和表征Not 4修饰的后果 基因调控蛋白的研究不仅将揭示复合物的新功能， 理解蛋白质泛素化在转录和DNA修复中的重要性。 基因表达缓冲是一种新的保守现象，在这种现象中， mRNA的合成和降解发生在对刺激的反应中，以维持mRNA的相似水平以“平衡”。 的反应。Ccr 4-Not与此过程有关，因为它控制合成和 破坏mRNA。缓冲的分子基础是未知的，其背后的机制是 在激烈的辩论中。我们已经绘制了与Ccr 4-Not相关的mRNA， 应激条件下，这揭示了Ccr 4从组成型mRNAs到由 氧化应激，这可能是缓冲的关键组成部分。主题2将揭示什么占 通过识别控制这一过程的顺式和反式因子， 反应，探索两种细胞去腺苷酸酶之间的相互作用，并确定蛋白质的变化， 在应激期间经历缓冲的mRNA的组成和定位。这些研究将确定 应激反应中mRNA靶向的决定因素，并揭示其背后的分子机制 基因表达缓冲。

Abstract: The goal of my research program is to understand how multifunctional protein complexes regulate gene expression, especially during stress responses. Our current focus is the yeast Ccr4-Not complex, which has been implicated in virtually all aspects of gene control, including transcription, mRNA decay, translational repression and protein ubiquitylation. The complex is it is highly conserved in all eukaryotes. Our laboratory has been a leader in understanding its role in transcription, especially RNA polymerase II elongation, employing multifaceted approaches including genetics, molecular biology, reconstitution biochemistry and genomics. Characterizing Ccr4-Not requires a flexible strategy and a willingness to move in new directions. This proposal will tackle two disparate processes under its control, namely the role of its ubiquitylation activity in gene expression and the mechanism of the reprogramming of Ccr4-Not mRNA targets during stress responses as a means to balance mRNA synthesis and decay. Protein destruction during transcription and DNA damage responses is an essential process. The Not4 subunit of the complex contains an E3 RING domain and controls ubiquitin-dependent destruction of proteins. We have shown that Ccr4-Not associates with elongation complexes and, recently, that it regulates the destruction of RNAPII after DNA damage. Theme 1 will identify novel targets of Not4 using global proteomics, identify the sites of modification and use molecular genetics and biochemistry to determine the consequences of ubquitylation on the protein’s function. Identifying and characterizing the consequences of Not4 modification of gene regulatory proteins will not only reveal novel functions of the complex, but lead to a greater understanding of the importance of protein ubiquitylation in transcription and DNA repair. Gene expression buffering is a novel conserved phenomenon where reciprocal changes in the rates in mRNA synthesis and degradation occur in response to stimuli to maintain similar levels of mRNAs to “balance” the response. Ccr4-Not has been implicated in this process because it controls both the synthesis and destruction of mRNAs. The molecular underpinnings of buffering are unknown and the mechanism behind it is under intense debate. We have mapped the mRNAs associated with Ccr4-Not under resting and oxidative stress conditions, which revealed extensive redistribution of Ccr4 from constitutive mRNAs to those induced by oxidative stress, which is likely a key component of buffering. Theme 2 will reveal what accounts for the reprogramming of the decay machinery during stress by identifying the cis- and trans- factors that control this response, explore the interplay between the two cellular deadenylases and identify changes in protein composition and localization of mRNAs undergoing buffering during stress. These studies will identify the determinants of mRNA targeting during stress responses and undercover the molecular mechanism behind gene expression buffering.