Naslov
Transfer RNA domains responsible for SerRS:tRNASer recognition and serylation in yeast and in methanogenic archaea

Autori
Jarić, Jelena ; Gruić-Sovulj, Ita ; Bilokapić, Silvija ; Dulić, Morana ; Weygand-Đurašević, Ivana

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
21st Internatinal tRNA Workshop, tRNA - 2005 / - Bangalore, 2005

Skup
TRNA 2005, 21st Internatinal tRNA Workshop

Mjesto i datum
Bangalore, Indija, 02.12.2005. - 07.12.2005

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
methanogenic archaea; yeast; SerRS:tRNASer recognition; tRNASer identity elements

Sažetak
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 have better insight into the mechanism of tRNASer serylation in methanogenic archaea, three Methanococcus maripaludis tRNASer isoacceptors were produced in vitro and subjected to aminoacylation with homologous M. maripaludis SerRS enzyme. Isoacceptor tRNASerMmGCT was the prefered substrate of M. maripaludis SerRS as shown by aminoacylation and gel retardation assay. When tRNASerMm isoacceptors were tested for charging by heterologous yeast SerRS, only tRNASerMmGCT showed activity at very low level. To improve charging with yeast SerRS and to locate domains of tRNASer responsible for recognition with M. maripaludis and yeast SerRS, particular regions of yeast tRNASer1 were introduced into the M. maripaludis tRNASerMmGCT framework. Kinetic analysis in the combination with noncovalent complex formation studies showed that productive recognition in yeast is achieved through the interaction of SerRS with variable and acceptor region of tRNASer. We propose that interaction with longer archaeal variable arm drives the formation of stable noncovalent complex in the presence of noncognate variable arm, but proper positioning of the acceptor stem is still not achieved. Furthermore, experiments of ATP-PPi exchange revealed that acceptor and variable domains are involved in the optimization of the serine binding site in yeast. Embedding of yeast anticodon stem and loop into tRNASerMmGCT almost completely abolished transcript serylation by M. maripaludis SerRS. This strongly suggests that anticodon region is an important identity domain in M. maripaludis. Contrary to yeast enzyme, it seems that the positioning of the acceptor stem in methanogenic archaea is not directly governed by interaction between variable arm of tRNA and SerRS, as shown by gel mobility shift and kinetic analysis. Noncovalent complexes between M. maripaludis enzyme and chimeric tRNAs, bearing one base pair shorter yeast variable arm, were not stable enough to be detected on the native PAGE. The interaction was detected only after covalent stabilization. Moreover, yeast enzyme showed the same covalent complex formation pattern with tRNASerMmGCT as with cognate tRNASer1, leading to the conclusion that the anticooperative binding of tRNA is conserved even with the noncognate tRNA.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA