T SPECIFIC DNA BINDING PROPERTIES OF A HUMAN PROTEIN

人类蛋白质的特定 DNA 结合特性

• 批准号: 2734707
• 负责人: RAYMOND REEVES
• 金额: $ 20.87万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734707
• 关键词: DNA footprinting; chemical binding; complementary DNA; cytokine receptors; gene induction /repression; genetic promoter element; human genetic material tag; interleukin 2; intermolecular interaction; nonhistone nucleoprotein; nucleosomes; polymerase chain reaction; site directed mutagenesis; tissue /cell culture

DESCRIPTION: The HMG-I(Y) group of mammalian "high mobility group" (HMG) chromatin proteins are in vivo regulators of gene expression and founding members of a new class of nonhistone proteins called 'architectural transcription factors'. The investigator has demonstrated in human T cells that HMG-I(Y) proteins participate, along with transcription factors NF-kB, Elf-1 and SRF, in the initial in vivo transcription induction of the interleukin-2 receptor a-chain gene (IL-2Ra), an essential controlling step in mounting an effective immune reaction. The goals of the proposed research are to elucidate, at the molecular/mechanistic level, how different regions of the HMG-I(Y) protein function in the transcriptional regulation of the IL-2Ra gene promoter. The investigator has produced a large number of both variant HMG-I(Y) proteins and mutant IL-2Ra promoter DNAs for use in both in vitro and in vivo investigations of the protein-DNA complexes formed on the IL-2Ra promoter during transcriptional activation. The Specific Aims of the research are to use these mutant proteins and promoter DNAs to: 1) Determine the relative importance of different peptide domains of the HMG-I(Y) protein in the formation of a looped, stereospecific activation complex on the promoter of the human IL-2Ra gene and investigate the molecular events involved in such complex formation in vitro and in vivo. 2) Determine the effects of site-specific HMG-I(Y) protein phosphorylations on protein-protein and protein-DNA interactions during promoter complex formation in vitro and in vivo. 3) Investigate the role played by HMG-I(Y) protein-induced alterations of the nucleosomal chromatin structure of the IL-2Ra promoter region during gene transcriptional activation in vitro and in vivo. A variety of extremely sensitive and quantitative biochemical, biophysical, and biological techniques will be employed to analyze the ability of mutant HMG-I(Y) proteins to participate in IL-2Ra promoter complex formation and function. Results from these experiments will contribute significant new information concerning the regulated expression of the IL-2Ra gene, one of the rate limiting steps in T cell activation during induction of the immune response.

描述：哺乳动物高迁移率族(HMG)的HMG-I(Y)群 染色质蛋白是基因表达和建立的体内调节因子 一类新的非组蛋白的成员，称为‘建筑’ 转录因子。研究人员已经证明，在人类T细胞中 HMG-I(Y)蛋白与转录因子NF-kB一起参与， ELF-1和SRF，在体内转录的初始诱导 白细胞介素2受体a链基因(IL-2ra)--一个重要的调控步骤 在建立有效的免疫反应方面。建议的目标是 研究是要在分子/机制层面上阐明 HMG-I(Y)蛋白在转录调控中的功能区域 IL-2ra基因启动子。调查员拿出了一大批 变异型HMG-I(Y)蛋白和突变型IL-2ra启动子DNA在临床中的应用 蛋白质-DNA复合体的体内外研究 转录激活过程中对IL-2ra启动子的影响。具体目标 研究的目的是使用这些突变蛋白和启动子DNA来：1) 确定不同多肽结构域的相对重要性 HMG-I(Y)蛋白在形成环状、立体特异性激活中的作用 人IL-2ra基因启动子上的复合体 分子事件参与了体外和体内这种复合体的形成。 2)确定位点特异性HMG-I(Y)蛋白磷酸化的影响 启动子复合体中蛋白质-蛋白质和蛋白质-DNA相互作用的研究 在体外和体内形成。3)调查HMG-I所起的作用(Y) 蛋白质诱导的核小体染色质结构的改变 IL-2ra启动子区域在体外基因转录激活中的作用 在活体内。各种极其灵敏和定量的生化， 生物物理学和生物技术将被用来分析 突变型HMG-I(Y)蛋白参与IL-2ra启动子的能力 复杂的形态和功能。这些实验的结果将 提供有关受调控表达的重要新信息 IL-2ra基因，T细胞活化的限速步骤之一 在免疫反应的诱导过程中。