engleski

tRNA-dependent amino acid discrimination by yeast seryl-tRNA synthetase

We have previously observed that yeast cytoplasmic seryl-tRNA synthetase (SerRS) displays a higher affinity toward serine in the overall aminoacylation assay than in the amino acid activation reaction lacking tRNA (1). The ability of tRNASer to influence the catalytic properties of SerRS in the activation of serine was demonstrated by pyrophosphate exchange conducted in the absence and presence of non-chargeable yeast tRNASer analogues. Two analogues, tRNASer oxidized at 3’-terminal adenosine (tRNASerox) and tRNASer truncated at 3’-end (tRNASerCC), decreased the KM value for serine and showed no effect on the KM value for ATP. In the presence of 500 nM tRNASerCC the KM value for serine in the pyrophosphate exchange reaction decreased ten-fold and became very close to the KM value determined in the overall aminoacylation. Heterologous E. coli tRNASer was not capable of inducing the complete conformational effect; the KM value for serine was less significantly decreased in the presence of E. coli tRNASer than of cognate yeast tRNASer. E. coli tRNASer was activated seryl-adenylate formation leading to 2.5 times higher kcat values in the PPi-exchange reaction. Even though tRNASer analogues slightly decreased the kcat values, possibly because the 3’-CCAox and 3’-CC ends interfere with the accessibility of the active site, the results presented suggest that the SerRS·tRNA non-covalent complex is catalyticaly more efficient in seryl-adenylate formation than non-complexed SerRS. Yeast SerRS was able to slightly misactivate threonine, but in the presence of tRNASerCC the formation of threonyl-adenylate was decreased at all threonine concentrations used, as confirmed by the ATP-PPi exchange assay. In summary, we present kinetic evidence that non-covalent interactions between SerRS and tRNASer induce a conformational change of the seryl-adenylate active site, leading to a conformation with higher affinity toward serine and with an increased turnover number in seryl-adenylate formation. The serine binding site is optimized in a tRNA-dependent manner, making misactivation of threonine less likely and less efficient. These results confirm the importance of non-covalent interactions between SerRS and tRNASer in maintaining overall fidelity of the aminoacylation reaction. (1) Lenhard, B. et al., J. Biol. Chem., 272 (1997) 1136.

aminoacyl-tRNA synthetases; tRNA; macromolecular complexes

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