GENE SELECTION BY MULTIPLE EUKARYOTIC RNA POLYMERASES

通过多种真核RNA聚合酶进行基因选择

• 批准号: 3271212
• 负责人: Marvin R. Paule
• 金额: $ 14.11万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-04-01 至 1990-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3271212
• 关键词: Acanthamoeba; DNA; DNA binding protein; DNA directed RNA polymerase; DNA footprinting; Saccharomyces; affinity chromatography; antibody; binding proteins; crosslink; electron microscopy; eukaryote; fungal genetics; gel electrophoresis; genetic transcription; immunochemistry; microorganism culture; protein biosynthesis; ribosomal DNA; ribosomal RNA; species difference

This is a proposal to study the mechanism of rRNA transcription and its regulation in eukaryotes. An in vitro transcription system made up of cloned rDNA, homogeneous RNA polymerase I (RNAP I) and an auxilliary protein (transcription initiation factor(s)-TIF) is being utilized: (1) to determine the number of TIFs, and to purify them to homogeneity, (2) to determine in greater detail the role played by the TIF in initiation of transcription, (3) to determine how the TIF and RNAP I interact with each other and with the template and (4) to describe kinetically the events involved in the assembly of the initiation complex. DNA footprinting and chemical modification-protection experiments will be used to determine protein-DNA contacts. Chemical cross-linking experiments will be used to determine protein-protein interactions. Kinetic measurements will be used to determine the kinetic mechanism of initiation. Monoclonal antibodies to the RNAP I subunits and the TIF(s) will be produced to aid in the analysis. Transcription of rRNA gene expression is developmentally regulated in Acanthamoeba by modification of RNAP I. Experiments to identify the chemical nature of the modification and the enzyme catalyzing it are proposed. Exactly which step in the process of initiation is affected by the modification is also to be studied utilizing the techniques proposed to study the mechanism of initiation.

这是研究rRNA转录及其调控机制的一项建议