Reconstitution of Transcription Using TATA-less Promoters

项目摘要 真核生物中的基因转录是由RNA聚合酶II（Pol II）完成的，这是六种通用转录酶。 因子（GTF）和基因特异性激活物、阻遏物和共调节物。转录失调和 对发育、环境和其他信号的不适当反应可能导致疾病。 转录开始于GTF对启动子的识别。以前的研究集中在 TATA盒结合蛋白（TBP）与启动子中的TATA盒的缔合。这些研究导致了 功能性前起始复合物（PIC）的完整组装和结构表征， Pol II和包含TATA启动子上的TBP的GTF。然而，80%的基因启动子 是"TATA-少"的，缺乏共有TATA序列。无TATA启动子需要多亚基TFIID 除了TBP之外，还有十三到十四个亚基的复合物，而不是单独的TBP。几乎没有，如果有的话， TATA-少启动子的TFIID依赖性转录在体外以相同的起始模式启动的证据 如在体内。事实上，在酵母酿酒酵母，重要的遗传研究，准确地启动 TATA-少启动子在体外的TFIID依赖性转录从未被观察到。无TATA的研究 基因转录通常用裸DNA模板进行。最近的研究表明， 然而，以染色质形式从酵母中分离的基因产生高得多的转录水平， 此外，转录起始位点的模式与体内发生的模式更加相似。的 这一提议的动机是这样的假设，即准确地启动TATA的TFIID依赖性转录， 较少的启动子需要染色质模板。 为了解决这个假设，我们将分离S。无TATA的RPS5核糖体编码蛋白 基因以染色质的形式在体内组装，并在体外与纯化的蛋白质一起转录。我们的首要目标是 通过定量蛋白质组学分析，鉴定与RPS5基因在体内相关的蛋白质。 RPS5染色质。然后，我们将用这些蛋白质重建RPS5染色质的转录， TFIID和许多蛋白质先前显示为在体外转录含TATA的基因所需的。 我们进一步的目标是使用在TFIID上组装的核小体来重建TFIID-核小体复合物。 体外RPS5启动子。这些实验将确定分子之间的关联要求 TFIID和TATA-少启动子，并导致通过冷冻电子技术对含有TFIID的PIC的结构进行了阐明 显微镜

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.