Reconstitution of Transcription Using TATA-less Promoters

使用无 TATA 启动子重建转录

• 批准号: 10311016
• 负责人: Jordan Taylor Feigerle
• 金额: $ 1.71万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10311016
• 关键词: Address; Biological Assay; Biology; Cancer Etiology; Chromatin; Complex; Consensus; Cryoelectron Microscopy; DNA; DNA Polymerase II; DNA-Directed RNA Polymerase; Development; Disease; Elements; Environment; Eukaryota; Gene Expression; Generations; Genes; Genetic Transcription; Genetic study; Goals; Histones; Human; Image; In Vitro; Knowledge; Laboratories; Lead; Life; Maps; Measures; Mediator of activation protein; Methods; Molecular; Motivation; Nucleosomes; Pattern; Positioning Attribute; Post-Translational Protein Processing; Procedures; Process; Proteins; Proteomics; Regulation; Regulator Genes; Reporting; Repressor Proteins; Research; Ribosomal Proteins; Saccharomyces cerevisiae; Signal Transduction; Site; Structure; Surface Plasmon Resonance; TAF1 gene; TATA Box; TATA-Box Binding Protein; Tail; Testing; Therapeutic Intervention; Trans-Activators; Transcription Initiation; Transcription Initiation Site; Transcription Process; Transcriptional Regulation; Work; Yeasts; experimental study; improved; in vivo; innovation; prevent; promoter; reconstitution; response; success; transcription factor

Project Summary Gene transcription in eukaryotes is performed by RNA Polymerase II (Pol II), six general transcription factors (GTFs) and gene specific activators, repressors and co-regulators. Misregulation of transcription and the resulting inappropriate response to developmental, environmental, and other signals can cause disease. Transcription begins with recognition of the promoter by the GTFs. Previous studies have focused on the association of the TATA-box binding protein (TBP) with the TATA-box in promoters. These studies have led to the complete assembly and structural characterization of a functional pre-initiation complex (PIC) composed of Pol II and the GTFs including TBP on TATA-containing promoters. However, eighty percent of gene promoters are “TATA-less”, lacking a consensus TATA sequence. TATA-less promoters require the multi-subunit TFIID complex with thirteen to fourteen subunits in addition to TBP, rather than TBP alone. There is little, if any, evidence of TFIID-dependent transcription of TATA-less promoters in vitro initiated at the same pattern of start sites as in vivo. Indeed, in the yeast Saccharomyces cerevisiae, important for genetic studies, accurately initiated TFIID-dependent transcription of TATA-less promoters in vitro has never been observed. Studies of TATA-less gene transcription have generally been performed with naked DNA templates. Recent work has shown, however, that genes isolated from yeast in the form of chromatin yield much higher levels of transcription, and moreover, the patterns of transcription start sites are much more similar to those occurring in vivo. The motivation for this proposal is the hypothesis that accurately initiated TFIID-dependent transcription of TATA- less promoters requires a chromatin template. To address this hypothesis, we will isolate the S. cerevisiae TATA-less RPS5 ribosomal protein-encoding gene in the form of chromatin assembled in vivo and transcribe with purified protein in vitro. Our first aim will be to identify the proteins associated with the RPS5 gene in vivo by quantitative proteomics analyses of the isolated RPS5 chromatin. We will then reconstitute transcription of RPS5 chromatin with these proteins, together with TFIID and the many proteins shown previously to be required for transcription of TATA-containing genes in vitro. We further aim to reconstruct a TFIID-nucleosome complex with the use of a nucleosome assembled on the RPS5 promoter in vitro. These experiments will define the molecular requirements for association between TFIID and a TATA-less promoter and lead to structural elucidation of a TFIID-containing PIC by cryo-electron microscopy.

项目摘要 真核生物中的基因转录由RNA聚合酶II（POL II），六个一般转录进行 因素（GTF）和基因特异性激活剂，复制品和共同调节剂。转录和 导致对发育，环境和其他信号的不当反应会导致疾病。 转录始于GTF对启动子的识别。以前的研究重点是 TATA-box结合蛋白（TBP）与启动子中TATA-box的关联。这些研究导致了 由功能性生成络合物（PIC）组成的完整组装和结构表征 POL II和GTF，包括含TATA的启动子的TBP。但是，80％的基因启动子 是“无塔塔”，缺乏共识tata序列。无塔塔的启动子需要多工资TfiID 除了TBP之外，还没有13至14个亚基复合物，而不是单独使用TBP。几乎没有（如果有的话） tfiID依赖性转录的证据是在同一开始模式下启动的无塔塔启动子的体外启动子的证据 如体内的站点。实际上，在酿酒酵母的酵母菌中，对于遗传研究很重要，可以准确地开始 从未观察到在体外的TFIID依赖性转录。无塔塔的研究 基因转录通常是用裸DNA模板进行的。最近的工作表明 但是，从染色质形式从酵母中分离出的基因产生了更高水平的转录水平，并且 此外，转录启动位点的模式与体内发生的模式更相似。这 该提议的动机是一个假设，即准确地启动了tata-的TFIID依赖性转录 较少的启动子需要染色质模板。 为了解决这一假设，我们将隔离酿酒酵母无RPS5核糖体蛋白质编码 染色质形式的基因在体内组装，并在体外用纯化的蛋白质转录。我们的第一个目标是 通过定量蛋白质组学分析，鉴定与RPS5基因相关的蛋白质 RPS5染色质。然后，我们将使用这些蛋白质重构RPS5染色质的转录 TFIID和许多蛋白质先前所示是在体外转录含Tata的基因所必需的。 我们进一步旨在通过使用在 RPS5启动子体外。这些实验将定义分子要求 TFIID和无TATA的启动子，并导致Cryo-Electron的含TFIID PIC的结构阐明 显微镜。