LYSYL-TRNA SYTHETASE AND APXN SYNTHESIS IN E COLI

大肠杆菌赖氨酰-tRNA合成酶(LysRS)系统由以下部分组成 两个差异调控的基因。一个叫Lyss的基因是结构性的，而且 第二个，Lysu，是可以诱导的。后者是一种已知的细胞应激基因， 其表达在热休克时被诱导。大肠杆菌中的氨基酰基- TRNA合成酶、LysRS和苯丙氨酰-tRNA合成酶(PHERS) 已经被证明是在体外合成最有效的 腺苷二核苷寡磷酸盐(ADO)，一类核苷酸家族 其中的原型是AP4A。ADO的胞内浓度 当大肠杆菌、其他细菌或真核细胞 易受热击、氧化剂或重金属等压力的 离子。重要的是要确定在大肠杆菌中是否每个LysRS的产物 基因和/或phers是体内合成的主要催化剂 ADO.在Lyss基因、Lysu基因或 Phes基因(Phers)以及一个双Lyss Lysu突变体和一个三倍Lyss Lysu突变体 Lyss Lysu PheS三重突变体现已在本实验室上市。这就是它 可以确定在细胞压力下，单个突变细胞，还是 具有双倍或三倍突变的菌株将具有正常的产量 ADO，或者如果每个LysRS基因产物和phers可能优先 合成ADO的一个子集。研究发现，细胞活力的丧失 压力与缺乏合成任何ADO会潜在地相关 为研究这些化合物的细胞作用指明了方向(S)。这 将是至关重要的，因为ADO在所有细胞中都是生成的，并且 被怀疑在DNA合成中起作用。此外，这项工作 有可能为一些人在体内建立一个基本的作用 非tRNA细胞氨酰化的氨酰基-tRNA合成酶 将通过测定之前和之前的集落形成单位来评估生存 在施加压力之后。这些核苷酸将用32P标记， 在应激之前，将在适当的时间采集样本 施加压力(热、氧化或重金属)。这个 核苷酸将通过二维薄层层析进行解析 (TLC)使用适当的标准，自动放射照相，切断板材和 通过闪烁计数进行定量。

The lysyl-tRNA synthetase (LysRS) system of Escherichia coli consists of two differentially regulated genes. One gene, lysS, is constitutive, and the second, lysU, is inducible. The latter is a known cell stress gene, and its expression is induced upon heat shock. Of the E. coli aminoacyl- tRNA synthetases, LysRS followed by phenylalanyl-tRNA synthetase (PheRS) have been shown in vitro to be the most efficient in synthesizing adenylylated dinucleoside oligophosphates (ADO), a family of nucleotides of which the prototype is AP4A. The intracellular concentration of the ADO rises markedly when E. coli, other bacteria, or eukaryotic cells are subjected to stresses such as heat shock, oxidizing agents, or heavy metal ions. It is important to determine if in E. coli the product of each LysRS gene, and/or PheRS are the predominant catalysts for the in vivo synthesis of the ADO. E. coli mutants defective in the lysS gene, the lysU gene, or the pheS gene (pheRS) as well a double LysS LysU mutants, and a triple LysS LysU PheS triple mutant are now available in this lab. This makes it feasible to determine whether upon cell stress, a singly mutant cell, or a strain with a double or triple mutation will have a normal output of ADO, or if each LysRS gene product and PheRS might preferentially synthesize a subset of ADO. The finding that a loss of cell viability upon stress correlates with a lack of synthesis of any ADO will potentially point the way, to studies on the cellular role(s) of these compounds. This would be of fundamental importance since ADO are made in all cells, and are suspected of having a role in DNA synthesis. Additionally this work has the potential to establish a fundamental in vivo role for some aminoacyl-tRNA synthetases other than for aminoacylation of tRNA cell survival will be assessed by determining colony forming units before and after application of a stress. The nucleotides will be labeled with 32P, prior to a stress, and samples will be taken at appropriate times upon application of the stress (heat, oxidation, or heavy metal). The nucleotides will be resolved by two-dimensional thin layer chromatography (TLC) using appropriate standards, autoradiographed, cut off the plate and quantitated by scintillation counting.