Initiation of mRNA decay in Bacillus subtilis

描述：(申请人提供)：我们的实验室试图了解 枯草芽孢杆菌mRNA腐烂的控制。对于革兰氏阳性细菌， 关于决定其功能的信使核糖核酸的特征，我们需要了解很多 半衰期，参与信使核糖核酸化的核糖核反应，基因 对其表达的调节进行核糖核酸编码。因为枯草杆菌基因组 没有几个主要核糖核酸酶基因的序列同源 在大肠杆菌中，完全不确定mrna的模型是否会衰退。 基于充分研究的大肠杆菌系统将属于枯草芽孢杆菌。 小的RNA分子将被用来探测B。 枯草杆菌：1)3‘外切核解的进入部位； 3)5‘端在细胞内的作用 测定信使核糖核酸半衰期。这些小的RNA分子被设计成 对它们的衰变的分析将开始阐明信使核糖核酸是如何实现周转的。 为了鉴定更多的核糖核酸酶基因(到目前为止已经克隆了三个)， 建议进行生物化学实验来分离几种预计将 参与了信使核糖核酸的衰变，即至少有一个额外的3‘到5’ 外切核糖核酸酶，一种可能的内切核糖核酸酶，以及多聚(A)聚合酶。一旦 这些蛋白质的特性是已知的，编码它们的基因将是 以研究其对信使核糖核酸衰变的影响。 加深对革兰氏阳性菌核糖核酸酶功能的认识 细菌，我们将研究BS-RNaseIII的功能，一个狭义的专一性 已被证明在枯草杆菌中必不可少的Enoribonuc租约。遗传 将采用各种手段来努力了解BS-RNase的作用 对生存能力至关重要。细菌BS-R.NaseIII活性控制的基础 将对该细胞进行研究，提供对核糖核酸酶基因的第一次了解 枯草杆菌的调控。

DESCRIPTION: (provided by applicant): Our laboratory seeks to understand the control of mRNA decay in Bacillus subtilis. For the Gram-positive bacteria, much needs to be learned about the features of an mRNA that determine its half-life, the ribonucleolytic reactions involved in mRNA decay, the genes that encode ribonuc the regulation of their expression. Since the B. subtilis genome does not have sequence homologues for several of the major ribonuclease genes of Escherichia coli, it is altogether uncertain whether models for mRNA decay based on the well-studied E. coli system will pertain to B. subtilis. Small RNA molecules will be used to probe three facets of mRNA turnover in B. subtilis: 1) the entry site for 3' exonucleolytic degradation; 2) endonucleolytic cleavage that initiates decay; and 3) role of the 5' end in determining mRNA half-life. These small RNA molecules are designed such that analysis of their decay will begin to clarify how mRNA turnover is achieved. To identify additional ribonuclease genes (three have been cloned thus far), biochemical experiments are proposed to isolate several proteins predicted to be involved in mRNA decay, i.e., at least one additional 3'-to-5' exoribonuclease, a putative endoribonuclease, and poly(A) polymerase. Once the identities of these proteins are known, the genes encoding them will be disrupted in order to study the effects on mRNA decay. To develop our understanding of ribonuclease function in the Gram-positive bacteria, we will study the function of Bs-RNase III, a narrow-specificity endoribonuc lease that has been shown to be essential in B. subtilis. Genetic means will be employed in an effort to understand the role of Bs-RNase Ill that is critical for viability. The basis for control of Bs-R.Nase III activity in the cell will be investigated, providing the first look at ribonuclease gene regulation in B. subtilis.