Biochemical & Genetic Analysis of Yeast SPN

描述(申请人提供)：酿酒酵母编码 两种蛋白质Sptl6和Pob3在真核生物中都高度保守， 包括人类。Sptl6和POB3起杂二聚体的作用，这个因子是 DNA复制和RNA转录都需要，因为细胞缺乏 正常蛋白质在这两个过程中都会显示错误。Sptl6-POB3对两者都有影响 每隔多久制作一次抄本，以及选定的确切启动地点。它 还能促进转录的延长。角色的广度令人惊讶 这一因素可以用一项简单的活动来解释： 调节核小体的性质。由于这些结构影响所有 真核生物基因组的区域，它们参与所有 涉及基因组，从建立转录的起始点到 复制，DNA和RNA聚合酶的进展，到包装 和基因组拷贝的分离。 Sptl6-POB3不同于其他真核生物的同源物，因为它缺乏一个 DNA结合基序。物理和遗传方法表明，它的功能 以及一种名为Nhp6的DNA结合蛋白。纯化的SPT 1 6-Pob3和Nhp6 在体外改变核小体的结构，从而改变其 电泳率并改变DNA在脑组织中的呈现 核小体。这一方案中的实验探索了变化的性质 Sptl6-Pob3-Nhp6(SPN)在核小体中的诱导，目的是了解 SPN如何改变基因组包装的这个基本单位的结构。 然后用遗传学方法测试SPN在细胞中的功能 描述复制和转录步骤的特定模型，可能是 由SPN推动。SPN活性减弱的影响随后在 揭示转录形成和复制起始的检测方法 SPN突变体中的复合体和中间产物的结构 伸长率。 Sptl6-POB3不像标准染色质重塑那样重新定位核小体 因子，但似乎是一种重组核小体的新型因子。 这些研究将阐明这种高度保守的因子在 调节染色质的基本成分的影响，并将 说明这一活动如何参与核酸的两个基本过程 代谢：转录和复制。

DESCRIPTION (provided by applicant): The yeast Saccharomyces cerevisiae encodes two proteins, Sptl6 and Pob3, that are each highly conserved among eukaryotes, including humans. Sptl6 and Pob3 function as a heterodimer, and this factor is required for both DNA replication and RNA transcription since cells lacking normal proteins display errors in both processes. Sptl6-Pob3 influences both how often transcripts are made and the precise site chosen for initiation. It also promotes elongation of transcripts. The surprising breadth of the roles of this factor can be explained by a single simple activity: the ability to modulate the properties of nucleosomes. Since these structures affect all regions of the eukaryotic genome, they are involved in all processes that involve the genome, from establishing initiation sites for transcription and replication, to the progression of DNA and RNA polymerases, to the packaging and segregation of genomic copies. Sptl6-Pob3 is unlike its homologs from other eukaryotes in that it lacks a DNA-binding motif. Physical and genetic methods indicate that it functions together with a DNA-binding protein called Nhp6. Purified Spt 1 6-Pob3 and Nhp6 alter the structure of nucleosomes in vitro in a way that changes their electrophoretc mobility and alters the presentation of the DNA in the nucleosome. Experiments in this proposal explore the nature of the changes induced in nucleosomes by Sptl6-Pob3-Nhp6 (SPN), with the goal of understanding how SPN changes the structure of this fundamental unit of genomic packaging. The function of SPN in cells is then addressed by using genetic methods to test specific models describing steps in replication and transcription that might be promoted by SPN. The effect of diminishing SPN activity is then examined in assays that reveal the formation of transcription and replication initiation complexes in SPN mutants, and the structure of intermediates formed during elongation. Sptl6-Pob3 does not reposition nucleosomes like a standard chromatin remodeling factor, but appears to be a new type of factor that reorganizes nucleosomes. These studies will elucidate activity of this highly conserved factor in modulating the effects of a fundamental component of chromatin, and will indicate how this activity participates in two basic processes of nucleic acid metabolism: transcription and replication.