Properties of the lysyl-tRNA synthetase gene and product from the extreme thermophile Thermus thermophilus.

Abstract:

: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.

journal_name

J Bacteriol

journal_title

Journal of bacteriology

authors

Chen J,Brevet A,Lapadat-Tapolsky M,Blanquet S,Plateau P

doi

10.1128/jb.176.9.2699-2705.1994

subject

Has Abstract

pub_date

1994-05-01 00:00:00

pages

2699-705

issue

9

eissn

0021-9193

issn

1098-5530

journal_volume

176

pub_type

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