THREONYL-TRANSFER-RNA SYNTHETASE FROM THERMUS-THERMOPHILUS - PURIFICATION AND SOME STRUCTURAL AND KINETIC-PROPERTIES

Threonyl-tRNA synthetase (ThrRS) has been isolated from an extreme the rmophile Thermus thermophilus strain HB8. The enzyme was purified to e lectrophoretic homogeneity by combinations of column chromatographies on DEAE-Sepharose, S-Sepharose, ACA-44 Ultrogel and HA-Ultrogel. Seven teen mg of purified enzyme were obtained from 1 kg of biomass. In para llel, purified aspartyl- and phenylalanyl-tRNA synthetases were obtain ed. The purified ThrRS is composed of two identical subunits with a mo lecular mass of about 77 000 (virtually the same as E coli ThrRS). The N-terminal sequence has been determined. The homology between the fir st 45 amino acid residues of ThrRS from T thermophilus and E coli is a bout 29%. A comparative study of tRNA(Thr) charging by ThrRS from E co li and T thermophilus reveals a similar efficiency of the reaction in both homologous systems. This efficiency remains unchanged for aminoac ylation of tRNA(Thr) from T thermophilus by the heterologous ThrRS fro m E coli, but decreases 700 times for aminoacylation of E coli tRNA(Th r) by ThrRS from T thermophilus.