Biochemical & Genetic Analysis of Yeast SPN

描述（由申请人提供）：酿酒酵母编码 两种蛋白质Spt 16和Pob 3，它们各自在真核生物中高度保守， 包括人类Sptl 6和Pob 3作为异二聚体起作用，并且该因子是 这是DNA复制和RNA转录所必需的，因为细胞缺乏 正常的蛋白质在这两个过程中都显示错误。Sptl 6-Pob 3影响两者 转录的频率和起始位点的精确选择。它 也促进转录物的延伸。令人惊讶的广泛作用， 这个因素可以用一个简单的活动来解释： 调节核小体的性质。由于这些结构影响到所有 在真核生物基因组的各个区域，它们参与所有的过程， 涉及基因组，从建立转录起始位点， 复制，DNA和RNA聚合酶的进展，包装 和基因组拷贝的分离。 Sptl 6-Pob 3与其来自其他真核生物的同源物不同，因为它缺乏一个 DNA结合基序。物理和遗传学方法表明它的功能 以及一种叫做Nhp 6的DNA结合蛋白。纯化的Spt 1 6-Pob 3和Nhp 6 在体外改变核小体的结构 抑制了DNA的流动性，改变了DNA在细胞中的表达。 核小体本提案中的实验探索了变化的本质 在核小体中由Sptl 6-Pob 3-Nhp 6（SPN）诱导，目的是了解 SPN如何改变基因组包装的基本单位的结构。 然后，通过使用遗传方法来测试SPN在细胞中的功能， 描述复制和转录步骤的特定模型， 由SPN推动。减少SPN活动的影响，然后检查 揭示转录和复制起始的形成的测定 复合物的SPN突变体，和中间体的结构形成的过程中， 伸长率 Sptl 6-Pob 3不像标准染色质重塑那样重新定位核小体 因子，但似乎是一种新型的因子，重组核小体。 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作用，并将 表明这种活性如何参与核酸的两个基本过程 新陈代谢：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.