LYSYL-TRNA SYTHETASE AND APXN SYNTHESIS IN E COLI

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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.