Lysyl-tRNA synthetase is a member of the class II aminoacyl-tRNA synthetases and catalyses the specific aminoacylation of tRNA(Lys). The crystal structure of the constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli has been determined to 2.7 A resolution in the unliganded form and in a complex with the lysine substrate. A comparison between the unliganded and lysine-bound structures reveals major conformational changes upon lysine binding. The lysine substrate is involved in a network of hydrogen bonds. Two of these interactions, one between the alpha-amino group and the carbonyl oxygen of Gly 216 and the other between the carboxylate group and the side chain of Arg 262, trigger a subtle and complicated reorganization of the active site, involving the ordering of two loops (residues 215-217 and 444-455), a change in conformation of residues 393-409, and a rotation of a 4-helix bundle domain (located between motif 2 and 3) by 10 degrees. The result of these changes is a closing up of the active site upon lysine binding.
The role of aminoacyl-tRNA synthetases in the in vivo synthesis of adenylylated bis(5'-nucleosidyl) tetraphosphates (Ap4N) was studied by measuring the concentration of these nucleotides in Escherichia coli cells overproducing lysyl-, methionyl-phenylalanyl-, or valyl-tRNA synthetase. Overproduction of each aminoacyl-tRNA synthetase (20-to 80-fold) was accompanied by a significant increase in intracellular Ap4N concentration (3-to 14-fold). As expected, nonadenylylated bis(5'-nucleosidyl) tetraphosphate concentration was not changed by synthetase overproduction. It was also verified that overproduction of an inactive methionyl-tRNA synthetase mutant did not modify Ap4N concentration. Ap4N accumulation during heat shock occurred in all strains studied.
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