NOVEL CONTROL MECHANISMS IN ENDOSPORE FORMATION

内孢子形成的新型控制机制

• 批准号: 2184521
• 负责人: ROBERT L SWITZER
• 金额: $ 14.41万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184521
• 关键词: Bacillus subtilis; DNA directed RNA polymerase; X ray crystallography; bacterial genetics; enzyme biosynthesis; enzyme structure; gene deletion mutation; genetic transcription; microorganism culture; mutant; nucleic acid structure; operon; pentosyltransferase; purine /pyrimidine metabolism; pyrimidine nucleotides; spores; transcription termination

This proposal is for research into the molecular details of regulation of the Bacillus subtilis pyrimidine nucleotide (pyr) biosynthetic operon by a novel autogenous transcription attenuation mechanism. The first gene of the 10 cistron pyr operon encodes a protein, PyrR, that acts to promote termination of transcription at three separate attenuation sites in the operon when exogenous pyrimidines are present in the growth medium. Experimental results obtained so far support a model for attenuation in which PyrR acts by binding in a UMP-dependent manner to specific sequences in pyr mRNA and favoring formation of a transcription terminator structure by preventing formation of a competing antiterminator structure. Experiments are proposed to test rigorously and to refine this model both in vitro and in vivo. An extraordinary feature of the PyrR regulatory protein is that it is also a uracil phosphoribosyltransferase with little sequence similarity to other uracil phosphoribosyltransferases. The PyrR protein, which has been purified to homogeneity and crystallized, will be fully characterized; its structure will be determined at high resolution. The relationship between the enzymatic activity of PyrR and its ability to regulate pyr transcription will be analyzed by biochemical and mutational studies. The primary and secondary structure of RNA sequences specifically bound by the pure PyrR protein will be studied. If cocrystallization can be achieved, the structure of a PyrR-RNA complex at high resolution will be determined. The regulation of pyr transcription in vitro by PyrR will be investigated using purified components. The secondary structures of pyr mRNA attenuators in the absence and presence of PyrR will be characterized. The regulation of attenuation at each of the three termination sites by pyrimidines and during sporulation will be investigated in vivo by overproduction of specific RNA fragments. Mutants in PyrR and in the attenuator sequences are being obtained by in vivo selection; these and mutants prepared by in vitro mutagenesis will be characterized. This research is focused on the fundamental nature of a novel and potentially very general regulatory mechanism, but it may also have eventual application of the development to new types of anti- microbial agents or to improvements in biotechnological production.

该提案旨在研究调节的分子细节 枯草芽孢杆菌嘧啶核苷酸（pyr）生物合成操纵子 一种新的自体转录减弱机制。第一个基因是 10 顺反子 Pyr 操纵子编码一种蛋白质 PyrR，其作用是促进 转录终止于三个独立的衰减位点 当生长培养基中存在外源嘧啶时操纵子。 迄今为止获得的实验结果支持衰减模型 PyrR 通过以 UMP 依赖性方式与特定序列结合来发挥作用 存在于pyr mRNA中并有利于转录终止子结构的形成 通过防止竞争性反终止子结构的形成。 建议进行实验来严格测试并完善该模型 体外和体内。 PyrR 监管的一个非凡特点 蛋白质的特点是它也是一种尿嘧啶磷酸核糖基转移酶，几乎没有 与其他尿嘧啶磷酸核糖基转移酶的序列相似性。吡咯 已纯化至均质并结晶的蛋白质将被 完全表征；其结构将以高分辨率确定。 PyrR 酶活性与其分解能力之间的关系 调节pyr转录将通过生化和突变进行分析 研究。 RNA序列的一级和二级结构具体 将研究纯 PyrR 蛋白的结合。如果共结晶可以 如果实现的话，高分辨率的 PyrR-RNA 复合物的结构将 被确定。 PyrR 在体外对 Pyr 转录的调控 使用纯化的成分进行研究。吡咯的二级结构 在 PyrR 不存在和存在的情况下，mRNA 衰减剂将 特点。三者中每一个的衰减调节 嘧啶和孢子形成期间的终止位点将是 通过特定 RNA 片段的过量产生进行体内研究。突变体 PyrR 和衰减子序列是通过体内获得的 选择；这些和通过体外诱变制备的突变体将是 特点。这项研究的重点是一个基本性质 新颖且可能非常普遍的监管机制，但它也可能 最终将开发成果应用于新型抗 微生物制剂或生物技术生产的改进。