Mechanism of yeast gene regulation

酵母基因调控机制

• 批准号: 10188562
• 负责人: KEVIN STRUHL
• 金额: $ 81.02万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10188562
• 关键词: 3' Untranslated Regions; Address; Affect; Area; Binding; Binding Proteins; Biochemical; Biological; Biological Phenomena; Biological Process; Chemicals; Chromatin Loop; Complex; DNA Sequence; Disease; Elements; Eukaryota; Evolution; Gene Expression; Gene Expression Regulation; Genetic; Genetic Transcription; Goals; Growth and Development function; Histone Acetylation; Histones; Human; Human Biology; Individual; Knowledge; Lead; Length; Light; Measurement; Measures; Mediating; Messenger RNA; Methodology; Molecular; Molecular Genetics; Mutation; Nature; Noise; Organism; Pattern; Physiological; Poly A; Polyadenylation; Prevention; Process; Protein Isoforms; Proteins; RNA; Regulation; Ribosomal Proteins; Role; Structure; TAF1 gene; Transcription Coactivator; Transcription Initiation; Transcription Process; Transcriptional Regulation; Variant; Work; Yeasts; base; cell growth; experimental study; functional genomics; human disease; improved; mRNA Stability; novel; promoter; protein protein interaction; recruit; response; transcription factor

PROJECT SUMMARY The mechanisms by which eukaryotes regulate gene expression are important for understanding many complex biological phenomena including human diseases. Prevention and treatment of such diseases have been and will continue to be improved by basic knowledge of gene regulation, especially because molecular mechanisms of transcriptional initiation are highly conserved in eukaryotic organisms ranging from human to yeast. This proposal will continue to investigate basic issues concerning molecular mechanisms of transcriptional regulation, polyadenylation, and mRNA stability in yeast, by combining molecular genetic, biochemical, functional genomic, and evolutionary approaches. Work in the first two sections will take advantage of our novel and recently developed methodologies for measuring half-lives, structure (DREADS via chemical probes), protein binding (CLIP-READS), and poly(A) length (A-READS) of individual mRNA 3' isoforms. First, in the area of polyadenylation, we will A) address the mechanisms for why polyadenylation is restricted to the 3' UTR. B) identify factors that are responsible for the wild-type poly(A) pattern, C) determine the factors and mechanistic basis for regulated polyadenylation during the diauxic shift (and perhaps other conditions), and D) elucidate 3'-isoform variation and regulation of poly(A) length. Second, for studies of mRNA stabilization/destabilization elements and half-lives of 3' isoforms and, we will A) perform RNA structural analysis during the degradation process, B) identify protein factors mediating the large differences in mRNA isoform stabilities C) perform directed genetic experiments to address how secondary structure affects mRNA stability, D) identify mRNA stabilization and destabilizing elements that differentially affected by environmental conditions, and E) identify factors important for regulated mRNA half-lives, which the goal of elucidating the mechanism of regulated mRNA stability. Third, we will address a variety of issues concerning transcriptional regulation including A) the nature of the transcriptional activator that coordinately regulates ribosomal protein gene expression via recruitment of TFIID, B) determining the mechanistic basis of why activator proteins do not function when bound downstream of or far away from the core promoter, C) DNA looping mechanisms, particularly the nature of the protein-protein interactions needed to form the loop and to stimulate transcription, and D) examining the role of histone acetylation in transcriptional regulation by generating non-acetylable derivatives of the 4 histones. Fourth, we will use a novel conceptual and experimental approach to distinguish biological function from biological noise that is based on a comparison of physiological responses, RNA and transcription factor binding profiles, and effects of mutations in yeast species of varying evolutionary distance. We will explicitly measure biological noise by making functional measurements of evolutionary irrelevant or random-sequence DNA in yeast. Overall, the proposal will answer fundamental questions about the interlinked processes of transcription, polyadenylation, and mRNA stability in a mechanistic and evolutionary framework.

项目总结 真核生物调节基因表达的机制对于理解许多 包括人类疾病在内的复杂生物现象。对此类疾病的预防和治疗 基因调控的基本知识已经并将继续得到改进，特别是因为分子 转录启动机制在真核生物中高度保守，从人类到 酵母。这项提案将继续研究与分子机制有关的基本问题 酵母中的转录调节、多聚腺苷酸化和mRNA稳定性，通过结合分子遗传， 生化、功能基因组和进化方法。前两个部分的工作将需要 我们新开发的测量半衰期、结构(DREADS通过 化学探针)、蛋白质结合(片段读取)和多聚(A)长度(A读取)。 异构体。首先，在多腺苷酸化方面，我们将A)阐述为什么多腺苷酸化是 仅限于3‘非编码区。B)确定导致野生型Poly(A)模式的因素，C)确定 调节多聚腺苷酸化的因素和机制基础在二位移位(可能还有其他 条件)和D)阐明聚(A)长度的3‘-异构体变异和调控。第二，研究 3‘亚型的mRNA稳定/失稳元件和半衰期，我们将A)执行RNA结构 在降解过程中的分析，B)确定调节mRNA差异较大的蛋白质因素 异构体稳定性C)进行定向的基因实验，以解决二级结构如何影响mRNA 稳定性，D)确定受环境影响不同的mRNA稳定和不稳定因素 条件，以及E)确定对调节的mRNA半衰期重要的因素，其目的是阐明 调节基因稳定性的机制。第三，我们将解决有关转录的各种问题 调节，包括A)协调调节核糖体蛋白的转录激活剂的性质 通过募集TFIID的基因表达，B)确定激活蛋白为什么不 当结合在核心启动子下游或远离核心启动子时起作用，C)DNA环机制， 尤其是形成环路和刺激转录所需的蛋白质-蛋白质相互作用的性质， 以及D)检验组蛋白乙酰化在转录调控中的作用 4个组蛋白的衍生物。第四，我们将使用一种新的概念和实验方法来区分 来自生物噪声的生物功能，基于生理反应的比较，RNA和 转录因子结合谱，以及不同进化距离的酵母物种中突变的影响。 我们将通过使进化或进化的功能测量不相关来明确测量生物噪声 酵母中的随机序列DNA。总体而言，该提案将回答有关相互关联的 在机制和进化框架中的转录、多聚腺苷酸化和信使核糖核酸稳定性的过程。