INITIATION OF MRNA DECAY IN BACILLUS SUBTILIS

本提案中的实验旨在了解 在革兰氏阳性细胞中的mRNA降解和mRNA加工 枯草芽孢杆菌。 细菌mRNA的快速更新是一种 基因表达调控的重要因素，并允许快速 微生物对变化的环境条件的适应。 衰变被认为是由在细胞中的内切核酸裂解开始的。 消息的主体，然后是3 '-5'核酸外切降解。 多核苷酸磷酸化酶（PNIPs）基因，一个主要的3' -to- B的5'核糖核酸外切酶。最近被克隆了， 含有该基因缺失的菌株（pnpA缺失菌株） 已经建成。 在pnpA缺失中缺少主要的3 '-至-5核糖核酸外切酶 菌株允许检测mRNA衰变的中间体 过程 我们建议，这些衰变中间体的特点，与 期望这将提供有关网站的新信息， 开始衰变的位置。 此外，缺乏 该菌株中的PNTR揭示了替代的3 '- Mn ~（2+）依赖性的to-5 ′核酸外切酶的基因克隆 对于这种核糖核酸酶的建议。 最后，PNTR缺陷型菌株 有几种突变表型，最明显的是对生长的敏感性， 四环素的存在。 我们假设这些突变体 表型是由于mRNA加工引起的表达改变。 另一个最近克隆的B。亚基基因编码Bs-RNase III， 窄特异性核糖核酸内切酶。 我们建议构建菌株 Bs-RNase III缺陷的细胞，以研究这种蛋白的功能。 RNA加工和基因表达中的酶。 其他 内切核糖核酸酶活性可能存在于B中。枯草芽抱杆菌w及 将建立一个体外系统来检测这些活动。

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.