Naslov
The role of N-terminal domain of seryl-tRNA synthetase from Methanosarcina barkeri in tRNA binding

Autori
Jarić, Jelena ; Bilokapić, Silvija ; Crnković, Ana ; Lesjak, Sonja ; Ban, Nenad ; Weygand-Durasevic, Ivana

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

Izvornik
HDBMB 2008 : book of abstracts / Strelec, Ivica ; Glavaš-Obrovac, Ljubica - Zagreb : Hrvatsko Društvo za Biotehnologiju, 2008, 50-50

ISBN
978-953-95551-2-0

Skup
HDBMB 2008 Congress of the Croatian Society of Biochemistry and Molecular Biology with International Participation

Mjesto i datum
Osijek, Hrvatska, 17.09.2008. - 20.09.2008

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Nije recenziran

Ključne riječi
seryl-tRNA synthetase ; tRNASer ; Methanosarcina barkeri ; macromolecular interaction ; nonsense suppression

Sažetak
Seryl-tRNA synthetase (SerRS) is a class II aminoacyl-tRNA synthetase that catalyzes serine activation and its transfer to cognate tRNASer. Methanogenic archaea possess unusual seryl-tRNA synthetase, evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. Previously reported crystal structure of the atypical Methanosarcina barkeri SerRS (mMbSerRS) reveals two idiosyncratic features of methanogenic-type SerRSs: a novel N-terminal tRNA- binding domain and an active site zinc ion. In order to gain an insight into the interaction between mMbSerRS and its cognate tRNASer, the bacterial SerRS-tRNASer and TyrRS-tRNATyr complex structures were used to model tRNA binding onto mMbSerRS. According to the model the long variable arm of the tRNA is positioned to interact with the highly conserved helices of the N-terminal domain of mMbSerRS. To determine the contribution of the amino acids in N-terminal domain involved in binding of tRNA, twelve mutated variants of mMbSerRS were produced. In each mutant one potentially important amino acid residue was replaced with alanine (R38A, R76A, R78A, K79A, K87A, K88A, Y89A, K90A, R94A, K141A, N142A and R143A). A significant drop in aminoacylation activity was detected for mutants K79A and R94A while mutant R76A was inactive in tRNA serylation. CD spectrometry analysis performed on the wild type enzyme and mutant R76A showed that the observed lack of tRNA charging with mutant R76A is not caused by protein misfolding. Furthermore, the activity of R76A, K79A, R94A and R143A mutants in amino acid activation was confirmed by ATP-PPi exchange assay. Surface Plasmon Resonace was used to determine dissociation constants (Kd) between tRNA and each mutant. Based on obtained results we conclude that amino acid R76 is crucial for tRNA binding while amino acids K79 and R94 have weaker impact but are presumably also involved in direct binding of tRNA. Side chains of R143 and K141 very likely indirectly influence complex formation between mMbSerRS and its cognate tRNA. The results have been verified in vivo by testing the ability of the mutants to suppress amber mutations in E. coli when co-expressed with the gene for cognate archaeal suppressor tRNA.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

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