AT SPECIFIC DNA BINDING PROPERTIES OF A HUMAN PROTEIN

AT 人类蛋白质的特定 DNA 结合特性

基本信息

批准号：
6179364
负责人：
RAYMOND REEVES
金额：
$ 21.97万
依托单位：
WASHINGTON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-08-01 至 2002-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6179364
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

项目摘要

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“高流动组”（HMG）的HMG-I（Y）组染色质蛋白是基因表达和基因的体内调节剂一类新的非组蛋白蛋白的成员称为“建筑转录因子”。研究者在人类T细胞中证明了 HMG-I（Y）蛋白与转录因子NF-KB一起参与 ELF-1和SRF，在初始体内转录诱导中白介素-2受体A链基因（IL-2RA），这是一个必不可少的控制步骤在安装有效的免疫反应时。提议的目标研究是在分子/机械水平上阐明如何不同的转录调节中HMG-I（Y）蛋白功能的区域 IL-2RA基因启动子。调查员产生了大量在变体HMG-I（Y）蛋白和突变体IL-2RA启动子DNA中均用于用于体外和体内研究的蛋白质-DNA复合物在转录激活过程中IL-2RA启动子上。具体目标这项研究是将这些突变蛋白和启动子DNA使用：1）确定不同肽域的相对重要性 HMG-I（Y）蛋白在形成循环的立体特异性激活中在人IL-2RA基因的启动子上复杂并研究在体外和体内参与这种复杂形成的分子事件。 2）确定位点特异性HMG-I（Y）蛋白磷酸化的影响在启动子复合物过程中蛋白质 - 蛋白质和蛋白-DNA相互作用体外和体内形成。 3）调查HMG-I（Y）扮演的角色蛋白质诱导的核小体染色质结构的改变基因转录激活在体外和体内。各种非常敏感和定量的生化，将采用生物物理和生物技术来分析突变HMG-I（Y）蛋白参与IL-2RA启动子的能力复杂的形成和功能。这些实验的结果将贡献有关受调节表达的重要新信息 IL-2RA基因的速率限制步骤之一在诱导免疫反应期间。