Biochemical & Genetic Analysis of Yeast SPN

生化

• 批准号: 6703123
• 负责人: Timothy G Formosa
• 金额: $ 28.26万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6703123
• 关键词: DNA binding protein; DNA replication; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; chromatin; chromatography; conformation; deoxyribonuclease I; eukaryote; functional /structural genomics; fungal genetics; fungal proteins; gel mobility shift assay; gene interaction; genetic mapping; genetic transcription; genome; immunoprecipitation; nucleosomes; protein structure function

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.

描述（由申请人提供）：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作为异二聚体起作用，这个因子是 由于细胞缺乏 DNA 复制和 RNA 转录所需的 正常蛋白质在这两个过程中都会出现错误。 Sptl6-Pob3 影响两者 转录本的制作频率以及起始选择的精确位点。它 还促进转录本的延伸。角色的惊人广度 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。由于这些结构影响所有 真核生物基因组的区域，它们参与所有过程 涉及基因组，从建立转录起始位点到 复制、DNA 和 RNA 聚合酶的进展、包装 和基因组拷贝的分离。 Sptl6-Pob3 与其他真核生物的同源物不同，因为它缺乏 DNA 结合基序。物理和遗传学方法表明其功能 与称为 Nhp6 的 DNA 结合蛋白一起。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 电泳迁移率并改变DNA在电泳中的呈现 核小体。该提案中的实验探索了变化的本质 由 Sptl6-Pob3-Nhp6 (SPN) 在核小体中诱导，目的是了解 SPN 如何改变基因组包装这一基本单位的结构。 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可能是 由 SPN 推广。然后检查 SPN 活性减弱的影响 揭示转录形成和复制起始的测定 SPN突变体中的复合物，以及过程中形成的中间体的结构 伸长。 Sptl6-Pob3 不会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。 这些研究将阐明这一高度保守因子的活性 调节染色质基本成分的作用，并将 表明该活性如何参与核酸的两个基本过程 新陈代谢：转录和复制。