Isolation, crystallization and preliminary X-ray analysis of the transamidosome, a ribonucleoprotein involved in asparagine formation.

ABSTRACT

Thermus thermophilus deprived of asparagine synthetase synthesizes Asn on tRNA(Asn) via a tRNA-dependent pathway involving a nondiscriminating aspartyl-tRNA synthetase that charges Asp onto tRNA(Asn) prior to conversion of the Asp to Asn by GatCAB, a tRNA-dependent amidotransferase. This pathway also constitutes the route of Asn-tRNA(Asn) formation by bacteria and archaea deprived of asparaginyl-tRNA synthetase. The partners involved in tRNA-dependent Asn formation in T. thermophilus assemble into a ternary complex called the transamidosome. This particule produces Asn-tRNA(Asn) in the presence of free Asp, ATP and an amido-group donor. Crystals of the transamidosome from T. thermophilus were obtained in the presence of PEG 4000 in MES-NaOH buffer pH 6.5. They belonged to the primitive monoclinic space group P2(1), with unit-cell parameters a = 115.9, b = 214.0, c = 127.8 A, beta = 93.3 degrees . A complete data set was collected to 3 A resolution. Here, the isolation and crystallization of the transamidosome from T. thermophilus and preliminary crystallographic data are reported.