Protein interactions and initiation by RNA polymerase I

蛋白质相互作用和 RNA 聚合酶 I 引发

• 批准号: 6968735
• 负责人: LAWRENCE I ROTHBLUM
• 金额: $ 21.83万
• 依托单位: GEISINGER CLINIC
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6968735
• 关键词: 3T3 cells; DNA directed RNA polymerase; DNA replication origin; SDS polyacrylamide gel electrophoresis; carcinogenesis; cell growth regulation; cell line; cytogenetics; enzyme induction /repression; gene expression; genetic transcription; high performance liquid chromatography; immunoprecipitation; liquid chromatography mass spectrometry; matrix assisted laser desorption ionization; phosphorylation; protein protein interaction; ribosomal DNA; ribosomal RNA; small interfering RNA; thin layer chromatography; transcription factor; western blottings

DESCRIPTION (provided by applicant): Understanding the mechanism of transcription of the genes that code for 45S pre-ribosomal RNA is essential if we are to understand both normal and abnormal growth processes, e.g. wound healing and neoplasia. Ribosome biogenesis, and therefore the expression of the ribosomal RNA genes, is coordinated with the rate of cell growth, and responds to a variety of signals, depending upon the cell type studied. The long-term objective of our research is to determine the mechanism(s) by which ribosomal RNA gene (rDNA) transcription is regulated. At least two trans-acting factors are required for accurate and efficient rDNA transcription, SL-1, and UBF. SL-1 is required for transcription while UBF activates transcription. Recent studies demonstrate that there are several ways in which rDNA transcription is regulated. One is the regulation of the ability of RNA polymerase I to initiate transcription. It has not been clear how many factors control the ability of pol I to initiate transcription. Two of the factors, Rrn3/TIF-IA and TFIC were believed to be the same factor. We demonstrated that they are two different factors. The yeast Rrn3 gene is essential for cell viability, and experiments in mammalian cells demonstrate that mammalian Rrn3 is essential for ribosomal gene transcription. However, there is considerable controversy concerning the role of Rrn3 in transcription, e.g. is it required for the recruitment of pol I to the rDNA promoter? Moreover, our data demonstrate fundamental differences between the mechanisms that regulate Rrn3 function in yeast and mammalian cells. Two of our goals focus on the determination of the role of Rrn3 in rDNA transcription and the mechanism(s) that regulate Rrn3 activity. Our third aim focuses on the role of PAF53, a second polymerase associated factor. Several lines of evidence suggest that PAF53 is an important component of the apparatus that regulates rDNA transcription. Antibodies to PAF53 block rDNA transcription, and the association of RNA polymerase I with PAF53 correlates with the rate of rDNA transcription. For example, PAF53 levels, but not core RNA polymerase I levels are reduced when NIH 3T3 cells are serum starved, while serum starvation causes the dissociation of PAF53 from RNA polymerase I in 3T6 cells. However, the role of PAF53 in rDNA transcription is yet to be defined.

描述（由申请人提供）：如果我们要了解正常和异常的生长过程，例如，了解编码 45S 前核糖体 RNA 的基因的转录机制至关重要。伤口愈合和肿瘤形成。核糖体生物发生以及核糖体 RNA 基因的表达与细胞生长速率相协调，并对各种信号做出反应，具体取决于所研究的细胞类型。我们研究的长期目标是确定核糖体 RNA 基因 (rDNA) 转录的调节机制。准确有效的 rDNA 转录至少需要两种反式作用因子：SL-1 和 UBF。 SL-1 是转录所必需的，而 UBF 则激活转录。最近的研究表明，rDNA 转录有多种调节方式。一是RNA聚合酶I启动转录能力的调节。目前尚不清楚有多少因素控制 pol I 启动转录的能力。其中两个因子 Rrn3/TIF-IA 和 TFIC 被认为是同一因子。我们证明它们是两个不同的因素。酵母 Rrn3 基因对于细胞活力至关重要，哺乳动物细胞中的实验表明哺乳动物 Rrn3 对于核糖体基因转录至关重要。然而，关于 Rrn3 在转录中的作用存在相当大的争议，例如pol I 募集至 rDNA 启动子是否需要它？此外，我们的数据证明了酵母和哺乳动物细胞中调节 Rrn3 功能的机制之间的根本差异。我们的两个目标集中于确定 Rrn3 在 rDNA 转录中的作用以及调节 Rrn3 活性的机制。我们的第三个目标集中于 PAF53（第二种聚合酶相关因子）的作用。多项证据表明 PAF53 是调节 rDNA 转录的装置的重要组成部分。 PAF53 抗体会阻断 rDNA 转录，并且 RNA 聚合酶 I 与 PAF53 的结合与 rDNA 转录速率相关。例如，当 NIH 3T3 细胞血清饥饿时，PAF53 水平降低，但核心 RNA 聚合酶 I 水平不降低，而血清饥饿会导致 3T6 细胞中 PAF53 与 RNA 聚合酶 I 解离。然而，PAF53 在 rDNA 转录中的作用尚未明确。