PROKARYOTIC CHROMATIN & TRANSCRIPTIONAL REGULATION

原核染色质

• 批准号: 3296387
• 负责人: ERNEST PETER GEIDUSCHEK
• 金额: $ 21.32万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3296387
• 关键词: DNA binding protein; DNA directed RNA polymerase; DNA replication; bacterial virus; chromatin; gene expression; genetic manipulation; genetic transcription; nuclear magnetic resonance spectroscopy; nucleic acid sequence; prokaryote; temperature sensitive mutant; virus assembly; virus genetics; virus protein

The proposed research program is in the general area of mechanisms for regulating the activity of genes. Its broad and practical implications are for understanding gene regulation in developing systems and infectious processes, and for precise interventions concerning gene activity. The particular system involves a bacterial virus, whose development is determined by a programmed sequence of gene expression. The virus codes for a chromatin-forming protein, called TF1, which is a homologue of the ubiquitous bacterial chromatin-forming proteins, called HU or type 11 DNA-binding proteins. The proposed research program pursues two lines of work. The first group of studies deals with the genetics, function, structure and DNA binding properties of TF1, by: 1) generating temperature-sensitive TF1 by mutagenesis targeting the entire TF1 gene, selectively; 2) analyzing defects of viral development, caused by the failure of TF1 function, at the molecular level; 3) analyzing structure- function relationships in TF1 by generating mutant TF1 with specific, appropriately chosen changes of amino acid sequence and analyzing the consequences of these changes for the affinity and specificity of DNA binding; 4) engaging in collaborations, involving X-ray crystallography and NMR spectroscopy, to determine the 3-dimensional structure of TF1 and of TF1-DNA complexes. A second group of in vivo and in vitro studies deals with the mechanism of selectively shutting off transcription at some, but not all, SP01 middle promoters by: 1) mapping and sequencing middle promoters of each regulatory subtype, seeking to detect distinctive conserved sequences that may correlate with one regulatory class or another: 2) utilizing the ability to clone in phage SP01 for analyzing promoter strength and regulation in vivo by molecular genetic methods; 3) analyzing overlapping late/middle specificity transcription initiation with appropriate in vitro systems and precise 5'-end mapping; 4) analyzing the occupancy of promoters by proteins in vivo; 5) examining effects of topoisomerase inhibitors on regulation at specific middle and late promoters; 6) examining the effect of DNA supercoiling on in vitro transcription at selected middle and late promoters; 7) analyzing the properties of virus-coded RNA polymerase-binding proteins in suitable in vitro systems. Two subsidiary projects are also proposed: one concerns hybrid TF1 molecules, and the other concerns gene expression at a viral gene regulation-specific promoter on a resident plasmid.

拟议的研究计划是机制的一般领域 用于调节基因的活性。 它广泛而实用 含义是理解基因调节在开发中 系统和传染性过程，以及精确的干预措施 关于基因活性。 特定系统涉及细菌病毒，其发育 由基因表达的编程序列确定。 这 病毒代码为染色质蛋白，称为TF1，是 无处不在的细菌染色质蛋白的同源物， 称为HU或类型11 DNA结合蛋白。 拟议的研究 计划追求两条工作。 第一组研究 处理遗传学，功能，结构和DNA结合 TF1的特性，作者：1）生成温度敏感的TF1 诱变有选择地靶向整个TF1基因； 2） 分析病毒发育的缺陷，是由 TF1功能，在分子水平上； 3）分析结构 - 通过与 特定的，适当选择的氨基酸序列变化和 分析这些变化对亲和力的后果和 DNA结合的特异性； 4）合作， 涉及X射线晶体学和NMR光谱，以确定 TF1和TF1-DNA复合物的3维结构。 一个 第二组体内和体外研究涉及 有选择地关闭转录的机制，但是 并非全部，SP01中间启动子：1）映射和测序中间 每个监管亚型的启动子，试图检测独特的 可能与一个调节类相关的保守序列 或另一个：2）利用在噬菌体SP01中克隆的能力 通过分子分析体内启动子强度和调节 遗传方法； 3）分析重叠的晚期/中间特异性 具有适当体外系统的转录启动， 精确的5'-end映射； 4）分析通过 体内蛋白质； 5）检查拓扑异构酶抑制剂的影响 在特定的中期和晚期发起人的监管； 6）检查 DNA超螺旋对体外转录的影响 选定的中间和晚期促进者； 7）分析特性 病毒编码的RNA聚合酶结合蛋白在适当的体外 系统。 还提出了两个辅助项目：一个问题 杂交TF1分子，其他涉及基因表达 病毒基因调节特异性启动子在驻留质粒上。