Naslov
Significance of the pre-transfer editing pathway in eukaryotic isoleucyl-tRNA synthetase

Autori
Šoštarić, Nikolina ; Cvetešić, Nevena ; Dulić, Morana ; Biluš, Mirna ; Gruić-Sovulj, Ita

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

Izvornik
24. hrvatski skup kemičara i kemijskih inženjera : Knjiga sažetaka / Ukić, Šime ; Bolanča, Tomislav - Zagreb : Hrvatsko društvo kemijskih inženjera i tehnologa (HDKI), 2015, 175-176

ISBN
978-953-6894-54-3

Skup
24. hrvatski skup kemičara i kemijskih inženjera

Mjesto i datum
Zagreb, Hrvatska, 21.04.2015. - 24.04.2015

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
isoleucyl-tRNA synthetase ; Saccharomyces cerevisiae ; pre-transfer editing

Sažetak
Cellular proteins arise at ribosomes whereby the genetic information given by mRNA is translated into the amino acid sequence. Amino acids participate in this process covalently attached at the 3’-end of the cognate tRNA. Ester bond formation between each amino acid and cognate tRNA pair is catalyzed by a specific aminoacyl- tRNA synthetase (aaRS). Accuracy of aminoacylation is crucial for the faithful protein synthesis. Certain aaRSs are incapable of distinguishing cognate and non-cognate amino acids with high accuracy in the synthetic reaction alone. To prevent errors in translation, these enzymes have developed complex hydrolytic proofreading activities. Proofreading may occur through hydrolysis of the aminoacyl-adenylate (pre- transfer editing) or hydrolysis of the aminoacyl- tRNA (post-transfer editing). Pre- transfer editing is typically catalysed by the enzyme’s synthetic active site in the absence of tRNA, however this activity may be significantly enhanced within the aaRS:tRNA complex (tRNA- dependent pre-transfer editing). Isoleucyl-tRNA synthetase (IleRS) is one of the error prone aaRSs, as it activates the non-cognate valine (Val) only 200 times less efficiently than the cognate isoleucine (Ile). This enzyme possesses a large appended hydrolytic domain which hosts the post-transfer editing activity. Besides that, Escherichia coli IleRS also exerts tRNA-dependent pre-transfer editing, an idiosyncratic activity of IleRS which may contribute up to 30 % of total IleRS proofreading activity. It was recently discovered that Tyr59 is the main determinant of tRNAdependent pre-transfer editing by E. coli IleRS. Intriguingly, Tyr59 was found to be conserved among prokaryotic IleRSs, but in the enzymes of eukaryotic origin it is replaced by either threonine or phenylalanine. This prompted us to question whether eukaryotic IleRSs lost the tRNA-dependent pre-transfer editing pathway. Saccharomyces cerevisiae IleRS (ScIleRS) was chosen as a model enzyme for kinetic characterization of the eukaryotic IleRS. It was shown that it discriminates valine and isoleucine similarly poor as the E. coli counterpart and therefore needs proofreading activity to preserve translational fidelity. To obtain high amounts of pure ScIleRS, its production was optimised in E. coli. Three variants of the fusion enzyme were overexpressed in E. coli under various experimental conditions. Extensive protocol for ScIleRS purification was developed to ensure high purity of the enzyme for kinetic characterization. We showed that ScIleRSs overexpressed in E. coli and in S. cerevisiae exhibit identical kinetic properties. These results justify heterologous overexpression of ScIleRS in E. coli in order to obtain large amounts of pure enzyme. Preliminary results with E. coli tRNAIle also suggest that ScIleRS does not exhibit tRNA-dependent pre- transfer editing. These results have intriguing evolutionary implications as tRNA-dependent pre- transfer editing may represent a remnant of primordial editing activity present only in the bacterial-type of IleRS. Purification of S. cerevisiae tRNAIle via complementary biotinylated oligonucleotides and verification of these results with the homologus tRNAIle is underway in our laboratory.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

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