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.

项目概要 真核生物中的基因转录是由 RNA 聚合酶 II (Pol II) 进行的，有六种通用转录 因子（GTF）和基因特异性激活剂、阻遏物和协同调节剂。转录失调和 对发育、环境和其他信号的不当反应可能导致疾病。 转录从 GTF 识别启动子开始。先前的研究主要集中在 TATA-box 结合蛋白 (TBP) 与启动子中的 TATA-box 的关联。这些研究导致 功能性预引发复合物 (PIC) 的完整组装和结构表征 Pol II 和 GTF，包括含有 TATA 的启动子上的 TBP。然而，百分之八十的基因启动子 是“TATA-less”，缺乏一致的 TATA 序列。无 TATA 启动子需要多亚基 TFIID 除了 TBP 之外，还含有 13 至 14 个亚基的复合物，而不是单独的 TBP。如果有的话，也很少， 无 TATA 启动子的 TFIID 依赖性转录在体外以相同的起始模式启动的证据 与体内一样的位点。事实上，在对遗传研究很重要的酿酒酵母中，准确地启动了 从未在体外观察到无 TATA 启动子的 TFIID 依赖性转录。无 TATA 的研究 基因转录通常使用裸DNA模板进行。最近的工作表明， 然而，以染色质形式从酵母中分离出来的基因产生了更高水平的转录，并且 此外，转录起始位点的模式与体内发生的更加相似。这 这个提议的动机是准确启动TATA-TFIID依赖性转录的假设 较少的启动子需要染色质模板。 为了解决这个假设，我们将分离出无 TATA 的酿酒酵母 RPS5 核糖体蛋白编码 基因以染色质形式在体内组装并在体外与纯化的蛋白质一起转录。我们的首要目标是 通过对分离的 RPS5 基因进行定量蛋白质组学分析，鉴定体内与 RPS5 基因相关的蛋白质 RPS5 染色质。然后我们将用这些蛋白质重建 RPS5 染色质的转录，以及 TFIID 和之前显示的许多蛋白质是含有 TATA 的基因体外转录所必需的。 我们进一步的目标是使用组装在 TFIID-核小体复合物上的核小体来重建 TFIID-核小体复合物。 RPS5 启动子体外。这些实验将定义之间关联的分子要求 TFIID 和无 TATA 启动子，并通过冷冻电子对含 TFIID 的 PIC 进行结构阐明 显微镜。