RESUMEN
The phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus is modularly composed of several different domains, some of which are not required for aminoacylation. In particular, the enzyme has the structural prerequisites for a DNA-binding protein. We demonstrate by gel retardation and competition experiments that the FRS specifically binds certain DNA sequences of the T. thermophilus genomic DNA. Although the implication of this finding is not yet understood, increasing evidence indicates an alternative function of this enzyme not related to aminoacylation. This might be a fundamental cellular process involved in cell proliferation which is related in bacteria and in humans.
Asunto(s)
ADN Bacteriano/metabolismo , Fenilalanina-ARNt Ligasa/metabolismo , Acilación , Aminoácidos/metabolismo , Unión Proteica , Especificidad por Sustrato , Thermus thermophilusRESUMEN
The alaS gene encoding the alanyl-tRNA synthetase (AlaRS) from Thermus thermophilus HB8 was cloned and sequenced. The gene comprises 2646 bp, corresponding to 882 amino acids, 45% of which are identical to the enzyme from Escherichia coli . The T. thermophilus AlaRS was overproduced in E.coli , purified and characterized. It has high thermal stability up to approximately 65 degrees C, with a temperature optimum of aminoacylation activity at approximately 60 degrees C, and will be valuable for crystallization. The purified enzyme appears as a dimer with a specific activity of 220 U/mg and k cat/ K M values of 118 000/s/M for alanine and 114 000/s/M for ATP. By genetic engineering a 53 kDa fragment of AlaRS comprising the N-terminal 470 amino acids (AlaN470) was also overproduced and purified. It is as stable as entire AlaRS and sufficient for specific aminoacylation of intact tRNAAla, as well as acceptor stem microhelices with a G3-U70, but not U3-A70, I3-U70 or C3-U70, base pair. The reduced binding strength of such microhelices to AlaN470 enabled, due to the resulting fast exchange of the microhelices between free and complexed states, preliminary NMR analyses of the binding mode and intermolecular recognition.
Asunto(s)
Alanina-ARNt Ligasa/metabolismo , Fragmentos de Péptidos/metabolismo , ARN de Transferencia de Alanina/metabolismo , Thermus thermophilus/enzimología , Alanina-ARNt Ligasa/química , Alanina-ARNt Ligasa/genética , Secuencia de Aminoácidos , Clonación Molecular , Escherichia coli/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Thermus thermophilus/genéticaRESUMEN
The contribution of entire domains or particular amino acid residues of the phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus to the interaction with tRNA(Phe) was studied. Removal of domain 8 of the beta subunit resulted in drastic reduction of the dissociation constant of the FRS x tRNA(Phe) complex. Neither the removal of arginine 2 of the beta subunit, which makes the only major contact between domains beta1-5 and the tRNA, nor the replacement of the conserved proline 473 by glycine had an influence on the aminoacylation activity of the FRS. Thus, the body comprising domains 1-5 of the beta subunit may not be essential for efficient aminoacylation of tRNA(Phe) by the FRS and rather be involved in other functions.
Asunto(s)
Fenilalanina-ARNt Ligasa/química , Thermus thermophilus/enzimología , Acilación , Adenosina Trifosfato/metabolismo , Estabilidad de Enzimas , Escherichia coli/genética , Cinética , Mutagénesis/genética , Fenilalanina-ARNt Ligasa/genética , Fenilalanina-ARNt Ligasa/metabolismo , Unión Proteica , Aminoacil-ARN de Transferencia/metabolismo , Eliminación de Secuencia/genética , TemperaturaRESUMEN
The phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus was overproduced in Escherichia coli. Three different promoter systems were used for the overexpression of the pheST genes: the tac, araB, and T7 promoters. Despite several attempts for improvement, the overproduction of the FRS was lower than that found with most of the other T. thermophilus genes. Nevertheless, enzyme amounts sufficient for biochemical and biophysical studies could be obtained more easily from the overproducing E. coli than from T. thermophilus, since at least fivefold higher specific FRS activity was present in the overproducing cells than in T. thermophilus. Also, a simple purification procedure was established. After heat treatment at 70 degrees C to remove thermolabile E. coli proteins, only three chromatographic steps, i.e., Q-Sepharose FF, hydroxyl apatite, and heparin-Sepharose chromatography, were necessary to obtain apparently homogeneous FRS. With a different plasmid construction we introduced six histidine residues at the N terminus of the alpha subunit. Thus, affinity chromatography on a nickel-chelate matrix can be used for the purification of FRS as well as for its mutant variants, which may be less stable than the native FRS and cannot be purified with heat treatment. We also cloned the pheST genes in a phagemid, which will enable mutagenesis studies and overexpression in a one-vector system without any subcloning steps.