PROPERTIES OF THE LYSYL-TRANSFER-RNA SYNTHETASE GENE AND PRODUCT FROMTHE EXTREME THERMOPHILE THERMUS-THERMOPHILUS

A DNA region carrying lysS, the gene encoding the lysyl-tRNA synthetas e, was cloned from the extreme thermophile prokaryote Thermus thermoph ilus VK-1 and sequenced. The analysis indicated an open reading frame encoding a protein of 492 amino acids. This putative protein has signi ficant homologies to previously sequenced lysyl-tRNA synthetases and d isplays the three motifs characteristic of class II aminoacyl-tRNA syn thetases. The T. thermophilus lysS gene was overexpressed in Escherich ia coli by placing it downstream of the E. coil beta-galactosidase gen e 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 a t 93 degrees C. Finally, lethal disruption of the lysRS genes of E. co li could not be compensated for by the addition in trans of the T. the rmophilus lysS gene despite the fact that this gene was overexpressed and that its product specifically aminoacylates E. coli tRNA(LyS) in v itro.