STUDIES ON THE CYCLIC AMP RECEPTOR PROTEIN OF E. COLI

大肠杆菌环AMP受体蛋白的研究

• 批准号: 3271229
• 负责人: JOSEPH S KRAKOW
• 金额: $ 13.88万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-04-01 至 1991-07-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3271229
• 关键词: DNA; Escherichia coli; affinity chromatography; antibody formation; bacterial genetics; binding proteins; carboxypeptidase; conformation; cyclic AMP; cyclic AMP receptors; gel electrophoresis; gel filtration chromatography; genetic transcription; hybridomas; immunologic techniques; laboratory mouse; lysine; monoclonal antibody; protein engineering; protein structure function; radiotracer; temperature sensitive mutant; tritium

The cAMP receptor protein (CRP) plays a key role in transcription of catabolite repressible operons as a positive regulatory factor via cAMP dependent DNA (promoter) binding. The proposed research involves an investigation of the role of CRP structure and function for cAMP and DNA binding. CRP has a domain structure in which the amino proximal region is involved in binding cAMP and subunit-subunit interaction while the carboxyl proximal region is involved in DNA binding. Polypeptide fragments and cores have been generated by limited proteolysis of CRP; these will be studied with regard to reconstitution of CRP activity, DNA binding and possible regulatory properties for in vitro lac and gal operon expression. Modification of CRP at the carboxyl terminus by carboxypeptidase will be carried out to determine the role of this region in DNA binding and CRP conformation. Chemical modification studies will be continued to determine effects on CRP structure and function. A temperature sensitive CRP mutant will be purified and characterized in terms of the ts lesion; revertants will be selected for study which show properties different from that of wild type CRP. Using hybridomas monoclonal antibodies will be prepared against CRP fragments. These monospecific antibodies will be used as probes of CRP conformation and function.

cAMP受体蛋白（CRP）在代谢产物的转录中起关键作用 阻遏型操纵子通过cAMP依赖性DNA作为正调控因子 （启动子）结合。 拟议的研究涉及调查的作用， CRP的结构和功能的cAMP和DNA结合。 CRP具有域 其中氨基近端区域参与结合cAMP的结构， 亚基-亚基相互作用，而羧基近端区域参与 DNA结合。 多肽片段和核心已经通过有限的 CRP的蛋白水解;将研究CRP的重建 活性，DNA结合和可能的调控特性，在体外乳糖和 gal操纵子表达。 CRP在羧基末端的修饰 将进行羧肽酶，以确定该区域在DNA中的作用 结合和CRP构象。 化学改性研究将继续进行 以确定对CRP结构和功能的影响。 温度敏感CRP 突变体将被纯化，并根据ts病变进行表征;回复突变体 将选择表现出不同于野生的特性的品种进行研究 CRP型。 使用杂交瘤制备抗CRP的单克隆抗体 片段 这些单特异性抗体将被用作CRP的探针 构象和功能。