engleski

Functional analysis of three tRNASer isoacceptors from archaeon Methanococcus maripaludis

Aminoacyl-tRNA synthetases aminoacylate tRNAs with cognate amino acids. The specificity of tRNA:synthetase recognition is crucial for fidelity of protein biosynthesis. Seryl-tRNA synthetase (SerRS) is a class II synthetase, comprising three signature motifs, characteristic for all class II enzymes. However SerRS enzymes from methane-producing Archaea Methanococcus jannaschii, Methanococcus maripaludis and Methanobacterium thermoautotrophicum share very limited identity (only about 16%) with both eukariotic- or bacteria-like representatives and have altered motif II. In order to biochemically characterize unusual SerRS enzyme from archaeon M. maripaludis, the overexpression of serS gene cloned in peT15 vector was achieved in E. coli. Enzyme was purified on HIS binding resin following FPLC Mono Q column. Synthetic genes for three tRNASer isoacceptors (anticodons GCT, TGA and GGA) from M. maripaludis were transcribed in vitro by T7 RNA polymerase. Renaturation studies made on tRNASer in vitro transcripts revealed that renaturation conditions highly depend on primary structure of tRNA. Since, in the aminoacylation assay tRNASerGCT has shown the highest acceptor activity, systematic renaturation analysis was performed for that transcript. tRNA samples were slowly renatured with different amount of Mg2+ added at different temperature and subjected to the native PAGE. Either additional bands of tRNA, possibly tRNA conformers or oligomers, or tRNA hydrolysis were observed. Only the fast renaturation (denaturation 5' at 80 °C, addition of 10 mM MgCl2 at 70 °C, cooling for 20' at RT) gave homogeneous sample of tRNASerGCT. Interestingly, even tRNA structures possessing altered mobility on the native PAGE seems to be properly recognized by SerRS in aminoacylation reaction. Noncovalent complex between SerRS and in vitro transcript tRNASerGCT was detected by the gel retardation assay at pH 7,0 in the presence of 10 mM Mg2+. Under the same conditions, noncovalent complexes between SerRS and tRNASerGGA and tRNASerTGA were not detected.

aminoacyl-tRNA synthetases; tRNA; macromolecular complexes; archaea

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