engleski

tRNA DOMAIN EXCHANGE GIVES NEW INSIGHT INTO THE tRNASer IDENTITY IN YEAST AND IN METHANOGENIC ARCHAEA

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 Escherichia coli. Enzyme was purified on histidine 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 these in vitro transcripts revealed that renaturation conditions highly depend on primary structure of tRNA. In the aminoacylation assay tRNASerGCT was the best substrate of M. maripaludis SerRS. Noncovalent complex between SerRS and in vitro transcript tRNASerGCT was detected by the gel retardation assay at pH 7.0 with 10 mM Mg2+. When M. maripaludis tRNASer isoacceptors were tested for charging by heterologous yeast and E. coli SerRS, the transcripts showed proper activity solely with E. coli enzyme. Yeast SerRS charged only tRNASerGCT transcript at very low level (100 pmol aminoacyl-tRNA/A260U). Therefore we decided to implant particular regions of yeast tRNASer into the M. maripaludis tRNASer molecule to improve charging with yeast SerRS and to locate tRNASer identity elements in M. maripaludis and in yeast. So, yeast acceptor stem, variable, D, TYC and anticodon arm, including their combinations like D-TYC and D-TYC-variable arm, were built one by one into the M. maripaludis tRNASerGCT isoacceptor. The chimeric molecules, bearing specified regions of yeast tRNASer in the M. maripaludis tRNASerGCT framework, were produced by in vitro procedure and subjected to kinetic and gel mobility shift analyses. Preliminary results suggest that insertions (17 and 17A) in the D-loop, characteristic of M. maripaludis tRNASers, are not significant identity elements of SerRS:tRNASer recognition in M. maripaludis. Time course curves pointed out acceptor and variable domain of tRNASer as important identity elements in both M. maripaldus and yeast.

aminoacyl-tRNA synthetases; tRNA

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