Existence of two distinct aspartyl-tRNA synthetases in Thermus thermophilus. Structural and biochemical properties of the two enzymes.
Two aspartyl-tRNA synthetases (AspRSs) were isolated from Thermus thermophilus HB8. Both are alpha2 dimers but differ in the length of their polypeptide chains (AspRS1, 68 kDa; and AspRS2, 51 kDa). Both chains start with Met and are deprived of common sequences to a significant extent. This rules out the possibility that AspRS2 is derived from AspRS1 by proteolysis, in agreement with specific recognition of each AspRS by the homologous antibodies. DNA probes derived from N-terminal amino acid sequences hybridize specifically to different genomic DNA fragments, revealing that the two AspRSs are encoded by distinct genes. Both enzymes are present in various strains from T. thermophilus and along the growth cycle of the bacteria, suggesting that they are constitutive. Kinetic investigations show that the two enzymes are specific for aspartic acid activation and tRNAAsp charging. tRNA aspartylation by the thermostable AspRSs is governed by thermodynamic parameters which values are similar to those measured for mesophilic aspartylation systems. Both thermophilic AspRSs are deprived of species specificity for tRNA aspartylation and exhibit N-terminal sequence signatures found in other AspRSs, suggesting that they are evolutionarily related to AspRSs from mesophilic prokaryotes and eukaryotes. Comparison of the efficiency of tRNA aspartylation by each enzyme under conditions approaching the physiological ones suggests that in vivo tRNAAsp charging is essentially ensured by AspRS1, although AspRS2 is the major species. The physiological significance of the two different AspRSs in T. thermophilus is discussed.
Becker
H D
HD
Unité Propre de Recherche 9002, Institut de Biologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, 67084 Strasbourg Cedex, France.
Reinbolt
J
J
Kreutzer
R
R
Giegé
R
R
Kern
D
D
D000595:Amino Acid Sequence; D001226:Aspartate-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015153:Blotting, Western; D002384:Catalysis; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D016415:Sequence Alignment; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D007527:Isoenzymes; D012346:RNA, Transfer, Amino Acyl; D001226:Aspartate-tRNA Ligase