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: 2396912
• 负责人: RAYMOND REEVES
• 金额: $ 21.12万
• 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/2396912
• 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）基团 染色质蛋白是体内基因表达的调节剂， 一种新的非组蛋白质类的成员，称为“建筑 转录因子。 研究人员已经在人类T细胞中证明 HMG-I（Y）蛋白沿着转录因子NF-κ B， Elf-1和SRF，在初始的体内转录诱导中， 白细胞介素-2受体α链基因（IL-2 Ra），一个重要的控制步骤 产生有效的免疫反应 拟议的目标 研究是为了阐明，在分子/机制水平上， HMG-I（Y）蛋白的区域在转录调节中起作用， IL-2 Ra基因启动子。 调查人员已经制作了大量的 变体HMG-I（Y）蛋白和突变型IL-2 Ra启动子DNA用于 形成的蛋白质-DNA复合物的体外和体内研究 在转录激活过程中IL-2 Ra启动子上。 具体目标 这项研究的目的是利用这些突变蛋白和启动子DNA：1） 确定不同肽结构域的相对重要性， HMG-I（Y）蛋白在环状立体特异性激活中的形成 复合物的人IL-2 Ra基因的启动子，并研究 在体外和体内参与这种复合物形成的分子事件。 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（Y）所起的作用 蛋白质诱导的核小体染色质结构的改变 IL-2 Ra启动子区在体外基因转录激活过程中的作用及 in vivo. 各种极其灵敏和定量的生化， 生物物理和生物技术将被用来分析 突变型HMG-I（Y）蛋白参与IL-2 Ra启动子的能力 复杂的形成和功能。 这些实验的结果将 提供有关受管制表达的重要新信息 IL-2 Ra基因，T细胞活化的限速步骤之一， 在诱导免疫反应期间。