engleski

tRNA identity domains in yeast and in methanogenic archaea

Aminoacyl-tRNA synthetases aminoacylate tRNAs with cognate amino acids. Their specificities are crucial for the 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 Methanocaldococcus jannaschii, Methanococcus maripaludis and Methanobacterium thermoautotrophicum share very limited identity (only about 16%) with both eukaryotic- or bacteria-like representatives and have altered motif II. In order to biochemically characterize unusual SerRS enzyme from archaeon M. maripaludis, methods for SerRS purification were developed. Synthetic genes for three tRNASer isoacceptors from M. maripaludis were transcribed in vitro by T7 RNA polymerase. tRNASerGCT was the best substrate of M. maripaludis SerRS as shown by aminoacylation and gel retardation assay. When tRNASer isoacceptors were tested for charging by heterologous yeast SerRS, the transcripts did not possess aminoacylation activity. Only tRNASerGCT transcript showed activity at very low level. 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. 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. Implantation of yeast anticodon stem and loop into M. maripaludis tRNASer almost completely abolished transcript serylation by M. maripaludis SerRS. This strongly suggests that anticodon region is an important identity element in M. maripaludis. Contrary to that, insertions (17 and 17A) in the D-loop, that are characteristic of M. maripaludis tRNASers, are shown not to be identity elements. Charging with yeast SerRS pointed out variable and acceptor domains as important identity elements in yeast with influence on tRNA affinity or SerRS catalytic efficiency, respectively.

tRNASer ; seryl-tRNA synthetase ; macromolecular compexes

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