INITIATION OF MRNA DECAY IN BACILLUS SUBTILIS

本提案中的实验旨在了解各方面 革兰氏阳性菌中的信使核糖核酸的降解和处理 枯草芽孢杆菌。细菌mRNA的快速周转是一种 调节基因表达的重要因素，并允许快速 微生物对变化的环境条件的适应。 腐烂被认为是从核内裂解开始的。 消息的正文，然后是3‘-5’核酸外切降解。 多核苷酸磷酸化酶(PNPase)基因是一种主要的3‘-脱氧核糖核酸 枯草杆菌的5‘外切核糖核酸酶，最近被克隆，并且一个 含有该基因缺失的菌株(pnpA缺失菌株) 已经建造好了。 PnpA缺失中缺少主要的3‘-5外核糖核酸酶 菌株可以检测到mRNA腐烂的中间产物 进程。我们建议用以下特征来描述这些腐烂的中间体 期望这将提供有关这些地点的新信息 腐烂开始发生的地方。此外，由于没有 该菌株中的PNPase揭示了替代的3‘- 依赖于Mn2+的To-5‘外切酶及其基因的克隆 对于这种核糖核酸酶提出了。最后，PNPase缺陷菌株 有几种突变表型，最明显的是对生长敏感 四环素的存在。我们假设这些突变体 表型是由于信使核糖核酸的处理改变了表达。 最近克隆的另一个枯草杆菌基因编码BS-RNaseIII，a 窄特异性内切核酸酶。我们建议构建菌株 在BS-RNaseIII中缺乏，以研究这一功能 RNA加工和基因表达中的酶。其他 枯草杆菌中可能存在核糖核酸酶活性，以及 将建立一个体外系统来检测这种活动。

The experiments in this proposal are aimed at understanding aspects of mRNA decay and mRNA processing in the Gram-positive bacterium Bacillus subtilis. Rapid turnover of bacterial mRNA is an important element in regulating gene expression, and allows quick adaptation of microorganisms to changing environmental conditions. Decay is thought to initiate with an endonucleolytic cleavage in the body of the message, followed by 3'-5' exonucleolytic degradation. The gene for polynucleotide phosphorylase (PNPase), a major 3' -to- 5' exoribonuclease of B. Subtilis, has recently been cloned, and a strain that contains a deletion of this gene (the pnpA deletion strain) has been constructed. The absence of the major 3'-to-5 exoribonuclease in the pnpA deletion strain has allowed detection of intermediates in the mRNA decay process. We propose to characterize these decay intermediates, with the expectation that this will provide new information about the sites at which initiation of decay occurs. Furthermore, the absence of PNPase in this strain has revealed the significance of an alternative 3'- to-5' exonuclease, which is Mn2+ dependent, and cloning of the gene for this ribonuclease is proposed. Finally, the PNPase deficient strain has several mutant phenotypes, most notably sensitivity to growth in the presence of tetracycline. We hypothesize that these mutant phenotypes are due to altered expression by mRNA processing. Another recently cloned B. Subtilis gene codes for Bs-RNase III, a narrow-specificity endoribonuclease. We propose to construct strains that are deficient in Bs-RNase III in order to study the function of this enzyme in RNA processing and gene expression. Other endoribonuclease activities are likely to be present in B. Subtilis, and an in vitro system will be set up to detect such activities.