engleski

Shuffling of Discrete tRNASer Regions Reveals Differently Utilized Identity Elements in Yeast and Methanogenic Archaea

Seryl-tRNA synthetases (SerRSs) from methanogenic archaea possess distinct evolutionary origin and show minimal sequence similarity with counterparts from bacteria, eukaryotes and other archaea. Here we show that SerRS from yeast Saccharomyces cerevisiae and archaeon Methanococcus maripaludis (ScSerRS and MmSerRS, respectively) display significantly different ability to serylate heterologous tRNASer. Recognition in yeast was shown to be more stringent than in archaeon. While cross-aminoacylation of M. maripaludis tRNASer (MmtRNASer) by yeast SerRS barely occurs, yeast tRNASer (SctRNASer) was shown to be a good substrate for heterologous MmSerRS. To investigate the contribution of different tRNA regions for the recognition by yeast and archaeal SerRS, chimeric tRNAs bearing separated domains of SctRNASer in MmtRNASer framework were produced by in vitro transcription and subjected to kinetic and gel mobility shift analysis with both enzymes. Generally, the recognition in M. maripaludis seems to be relatively relaxed toward tertiary elements of tRNASer structure and relies on the direct recognition of identity nucleotides. On the other hand, expression of tRNASer identity elements in yeast seems to be more sensitive toward surrounding sequence context. In both systems variable arm of tRNAwas recognized as a major identity region with a strong influence on SerRS:tRNA binding. Acceptor domain of SctRNASer was also shown to be important for serylation in yeast. We propose that cognate interactions between N-terminal domain of yeast SerRS and variable region of SctRNASer place the acceptor stem into the enzyme's active site and lead to increased affinity toward serine and efficient serylation of tRNA. The same effect was not observed in M. maripaludis. Unlike its yeast counterpart, MmSerRS forms only one type of covalent complex with MmtRNASer, regardless of the tRNA/SerRS molar ratio. Stoichiometry of the complex, one tRNA per dimeric SerRS, was revealed by mass spectrometry. Our studies indicate that different SerRS:tRNA recognition mode is utilized by these two systems.

tRNASer identity elements; SerRS; SerRS:tRNASer complexes; yeast; methanogenic archaea

nije evidentirano