Properties of the lysyl-tRNA synthetase gene and product from the extreme thermophile Thermus thermophilus.
A DNA region carrying lysS, the gene encoding the lysyl-tRNA synthetase, was cloned from the extreme thermophile prokaryote Thermus thermophilus VK-1 and sequenced. The analysis indicated an open reading frame encoding a protein of 492 amino acids. This putative protein has significant homologies to previously sequenced lysyl-tRNA synthetases and displays the three motifs characteristic of class II aminoacyl-tRNA synthetases. The T. thermophilus lysS gene was overexpressed in Escherichia coli by placing it downstream of the E. coli beta-galactosidase gene promoter on plasmid pBluescript and by changing the ribosome-binding site. The overproduced protein was purified by heat treatment of the crude extract followed by a single anion-exchange chromatography step. The protein obtained is remarkably thermostable, retaining nearly 60% of its initial tRNA aminoacylation activity after 5 h of incubation at 93 degrees C. Finally, lethal disruption of the lysRS genes of E. coli could not be compensated for by the addition in trans of the T. thermophilus lysS gene despite the fact that this gene was overexpressed and that its product specifically aminoacylates E. coli tRNA(Lys) in vitro.
Chen
J
J
Laboratoire de Biochimie, URA 240 du Centre National de la Recherche Scientifique, Ecole Polytechnique, Palaiseau, France.
Brevet
A
A
Lapadat-Tapolsky
M
M
Blanquet
S
S
Plateau
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D006358:Hot Temperature; D008250:Lysine-tRNA Ligase / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics*
D011994:Recombinant Proteins; D008250:Lysine-tRNA Ligase