NOVEL CONTROL MECHANISMS IN ENDOSPORE FORMATION

这项建议是为了研究调控的分子细节。 枯草杆菌嘧啶核苷酸(PYR)生物合成操纵子 一种新的自体转录衰减机制。人类第一个基因 10顺反子PYR操纵子编码一种蛋白质，PYR，其作用是促进 在三个不同的衰减点终止转录 外源嘧啶存在于生长介质中时的操纵子。 到目前为止所获得的实验结果支持一种衰减模型 哪些PyrR通过以依赖于UMP的方式与特定序列结合来发挥作用 并促进转录终止子结构的形成 通过防止形成竞争的反终止子结构。 建议进行实验，以严格测试并完善该模型 在体外和体内。PYRR监管机构的一个非凡功能 蛋白质是它也是一种尿嘧啶磷酸核糖基转移酶 序列与其他尿嘧啶磷酸核糖基转移酶的相似性。The PyrR 蛋白质，已经被提纯到均质并结晶，将被 完全表征；其结构将以高分辨率确定。 PYRR的酶活性与其降解能力的关系 将通过生化和突变来分析调节PYR转录 学习。RNA序列的一级和二级结构 将研究与纯净的PyrR蛋白结合。如果共结晶可以 实现了高分辨率的PyrR-RNA复合体的结构 要下定决心。PYR Will对PYR体外转录的调节作用 使用提纯的成分进行研究。PYR的二级结构 在没有和存在PyrR的情况下，mRNA衰减剂将 特色化的。三者中每一者的衰减规律 由嘧啶和产孢子过程中的终止位点将是 通过过量生产特定的RNA片段在体内进行研究。突变者 在吡咯受体中和在衰减器序列中通过体内获得 筛选；这些和通过体外诱变制备的突变体将被 特色化的。这项研究的重点是一个基本的性质 新颖且可能非常通用的监管机制，但它也可能 最终将该开发应用于新型防弹 微生物制剂或生物技术生产的改进。

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.