Threonyl-tRNA synthetase from Thermus thermophilus: Purification and some structural and kinetic properties

Crystallization of threonyl‐tRNA synthetase from Thermus thermophilus and preliminary crystallographic data

“…The molecular weight of the polypeptide chain of the overproduced TRS (75000) checked on SDS/PAGE is similar to that of the TRS purified from T. thermophilus [15]. By gel filtration on a Protein-Pack 300SW column under native conditions, TRS eluted as a globular protein of apparent molecular weight of 170 + 10 kDa (data not shown).…”

“…We found that the presence of 4% glycerol (v/v) in the elution buffer prevented protein aggregates and, therefore, improved the purification. The specific activity of the enzyme fractions obtained after the different purification steps could not be quantified with enough accuracy to establish a table of purification, since E. coli tRNA TM in the unfractionated tRNA we used in the test is poorly threonylated by ttTRS [15]. For this reason, the purification was followed by SDS/PAGE (Fig.…”

“…If necessary the enzyme was diluted in 20 mM Tris-C1 buffer, 30 mM KC1, 20 mM MgC12. The reaction was carried out at 37°C and the amount of [~4C]threonyl-tRNA formed at various incubation times was determined as described [15].…”

“…Furthermore, T. thermophilus is a thermophilic eubacterium close to E. coli. In particular, it has been shown that the extent of identity between homologous proteins and nucleic acids of the two species are generally high and that crossreactions between aaRS and tRNA occur [14,15]. An E. coli strain overproducing the thermostable TRS has been constructed.…”

Identity elements of Thermus thermophilus tRNAThr

“…Thermus thermophilus is an extremely thermophilic bacterium. According to a crossaminoacylation study of E. coli and T. thermophilus, ThrRS from E. coli can aminoacylate tRNA Thr from T. thermophilus as efficiently as homologous tRNA Thr, whereas tRNA Thr from E. coli is a poor substrate for ThrRS from T. thermophilus [21]. This result suggested a difference in the identity elements of tRNA Thr between mesophilic and thermophilic prokaryotes, although the tRNA Thr sequences are similar between E. coli and 7".…”