RESUMO
A nucleotide sequence of tRNA(Tyr) from the extreme thermophile Thermus thermophilus HB-27 living at 75 degrees C was determined. It is 86 nt long and shares a 52% homology with tRNA(Tyr) from Escherichia coli. A comparative analysis of the interaction sites of tRNA(Tyr) from T. thermophilus and E. coli with the cognate aminoacyl-tRNA synthetases was accomplished by the chemical modification and nuclease hydrolysis approaches. The tRNA(Tyr) was shown to interact with the cognate enzyme in the anticodon stem (on the 5'-side), in the anticodon, in the variable stem and loop (on the 5'-side), and in the acceptor stem (on the 3'-side). These regions are located in the variable stem of the L-form. It was demonstrated that, upon forming the complex E. coli tRNA(Tyr)-cognate synthetase, endonuclease V1 induces additional cleavages of phosphodiester bonds on the 3'-side of the anticodon stem and on the 5'-side of the T-stem. This implies that tRNA may change its conformation when it interacts with the enzyme.
Assuntos
Aminoacil-tRNA Sintetases/química , Escherichia coli/química , RNA Bacteriano/química , RNA de Transferência de Tirosina/química , Ribonucleases/química , Thermus thermophilus/química , Autorradiografia , Sequência de Bases , Escherichia coli/genética , Hidrólise , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Thermus thermophilus/genéticaRESUMO
Crystals have been obtained of threonyl-tRNA synthetase from the extreme thermophile Thermus thermophilus using sodium formate as a precipitant. The crystals are very stable and diffract to at least 2.4 A. The crystals belong to space group P2(1)2(1)2(1) with cell parameters a = 61.4 A, b = 156.1 A, c = 177.3 A.
Assuntos
Aminoacil-tRNA Sintetases/isolamento & purificação , Thermus/enzimologia , Treonina-tRNA Ligase/isolamento & purificação , Cristalização , Estabilidade Enzimática , Conformação Proteica , Difração de Raios XRESUMO
Crystals have been obtained of seryl-tRNA synthetase from the extreme thermophile Thermus thermophilus, using mixed solutions of ammonium sulphate and methane pentane diol. The crystals are very stable and diffract to at least 2 A. The crystals are monoclinic (space group P21) with cell parameters a = 87.1 A, b = 126.9 A, c = 63.5 A and beta = 109.7 degrees.
Assuntos
Aminoacil-tRNA Sintetases , Serina-tRNA Ligase , Thermus/enzimologia , Difração de Raios XRESUMO
A method for isolating tyrosyl-tRNA synthetase from Thermus thermophilus is described, including ammonium sulfate fractionation, chromatography on DEAE-sepharose, hydroxyapatite, heparin-sepharose and hydrophobic chromatography on Toyopearl HW-65. The yield of the purified enzyme was 1.6 mg per 1 kg of T. thermophilus cells. The enzyme is a dimer protein of the alpha 2 type with molecular weight of 100 kDa.