NOVEL CONTROL MECHANISMS IN ENDOSPORE FORMATION

内孢子形成的新型控制机制

• 批准号: 2392164
• 负责人: ROBERT L SWITZER
• 金额: $ 24.4万
• 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/2392164
• 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.

这项建议是为了研究调控的分子细节， 枯草芽孢杆菌嘧啶核苷酸（pyr）生物合成操纵子， 一种新的自体转录衰减机制。第一个基因 10个顺反子Pyr操纵子编码蛋白质PyrR，其作用是促进 转录终止在三个不同的衰减位点， 当生长培养基中存在外源嘧啶时， 到目前为止获得的实验结果支持衰减模型， PyrR通过以UMP依赖性方式结合特定序列发挥作用， 并有利于形成转录终止子结构 通过防止竞争性抗终止子结构的形成。 实验提出了严格的测试和完善这一模型， 在体外和体内。PyrR监管的一个非凡特征是 蛋白质的另一个特点是它也是一种尿嘧啶磷酸核糖基转移酶， 与其他尿嘧啶磷酸核糖基转移酶的序列相似性。PyrR 蛋白质，已被纯化至同质和结晶，将 充分表征;将以高分辨率确定其结构。 PyrR的酶活性与其抗肿瘤能力的关系 将通过生物化学和突变分析来调节pyr转录。 问题研究RNA序列的一级和二级结构 将研究由纯PyrR蛋白结合的蛋白质。如果共结晶可以 在高分辨率下，PyrR-RNA复合物的结构将 被确定。PyrR在体外对pyr转录的调控将 使用纯化的成分进行研究。Pyr的二级结构 在不存在和存在PyrR的情况下，mRNA衰减剂将是 表征了在三个区域中的每一个处的衰减的调节 终止位点和孢子形成期间， 通过过量产生特定RNA片段进行体内研究。突变体 在PyrR和衰减子序列中， 选择;这些和通过体外诱变制备的突变体将被 表征了这项研究的重点是一个基本的性质， 新的和潜在的非常普遍的调节机制，但它也可能 最后将其应用于新型的反 微生物剂或生物技术生产的改进。