engleski

Plant seryl-tRNA synthetases : fidelity and macromolecular recognition

Aminoacyl-tRNA synthetases (AARSs) catalyze the attachment of amino acids to their cognate tRNAs to establish the genetic code. To obtain the high degree of accuracy that is observed in translation, these enzymes must exhibit strict substrate specificity for their cognate amino acids and tRNAs. Many AARSs participate in macromolecular associations that improve their function or allow them to participate in various cellular processes, other than translation. Among eukaryotes, the recognition elements of tRNASer were analyzed in detail only in yeast and humans. To determine identity requirements in plant serine system, cross-species complementation test and aminoacylation assay were performed. Maize cytosolic SerRS (SerZMc) efficiently recognized bacterial and eukaryotic tRNAsSer indicating that SerZMc can accommodate various types of tRNASer structures. Genetic experiments using E. coli tyrosine-specific tRNA suppressor showed that the discriminator base G73 is crucial for recognition by SerZMc. The above data indicate that maize cytosolic SerRS has a broad tRNA specificity and a very low requirement for tRNA identity elements. Although SerZMc efficiently recognized bacterial tRNAsSer, which are similar to maize organellar tRNAsSer, GFP tagging experiments indicated its exclusive cytosolic localization. Since maize nuclear genome contains numerous organellar tRNASer genes, we hypothesize that some of them are expressed and their corresponding tRNAs are recognized by SerZMc in the cytosol. Fidelity of translation is important for normal cell functioning as well as for recombinant protein production of biotechnological importance in plant cytosol or chloroplasts. Therefore, we compared the fidelity of maize cytosolic and dually targeted organellar SerRS (SerZMo) with respect to amino acid recognition. Both enzymes weakly misactivated near-cognate amino acids threonine and cysteine, SerZMo being slightly more accurate enzyme compared to SerZMc. However, both enzymes were capable of hydrolytic editing implying their high overall accuracy. Thus far, macromolecular associations of plant AARSs were not reported. We used high throughput interaction technologies to determine protein partners of plant SerRS. Potential interactors identified in Y2H screen could not be confirmed in vitro, possibly due to transient nature of their interactions with SerRS. Preliminary data of TAP/MS experiments indicate various translation factors as putative interactors of plant SerRS.

aminoacyl-tRNA synthetase ; plant ; tRNA identity ; amino acid recognition ; editing ; protein interactions

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