Naslov
A single synthetic site residue modulates partitioning of pre- and post- transfer editing pathways in overall editing by isoleucyl-tRNA synthetase from Escherichia coli

Autori
Dulić, Morana ; Perona, John Joseph ; Gruić- Sovulj, Ita

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

Izvornik
HDBMB2014: The Interplay of Biomolecules, Abstract book / Katalinić, Maja ; Kovarik, Zrinka - , 2014

Skup
HDBMB2014: The Interplay of Biomolecules

Mjesto i datum
Zadar, Hrvatska, 24.09.2014. - 27.09.2014

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Nije recenziran

Ključne riječi
isoleucyl-tRNA synthetase; pre-transfer editing; proofreading; active site mutants

Sažetak
Isoleucyl-tRNA synthetase (IleRS) covalently attaches isoleucine to its cognate tRNAIle in a two-step reaction. First, isoleucine is activated by ATP yielding isoleucyl-adenylate, followed by the transfer of isoleucyl moiety to tRNA. IleRS also activates structurally similar noncognate valine and transfers it to tRNAIle. To maintain accuracy of translation, IleRS, as many other aminoacyl-tRNA synthetases, evolved a network of hydrolytic proofreading activities. Rapid deacylation of misacylated tRNA or post-transfer editing occurs in a separate editing domain dedicated to this activity alone. In contrast, hydrolysis of aminoacyl-adenylate or pre-transfer editing is in the most cases only a weak tRNA-independent activity. Rarely, as in IleRS, it is stimulated by the presence of tRNA. We have recently shown that both tRNA-independent and tRNA-dependent pre-transfer editing activities are in IleRS localized within the confines of the synthetic site. Furthermore, kinetic partitioning of valyl-adenylate between hydrolysis and the aminoacyl transfer reactions is determined by the ratio of their corresponding rates. In this work, we introduce an improved kinetic approach to distinguish editing reaction from aminoacylation, since both reactions contribute to AMP formation in the common editing assay. This approach revealed that tRNA dependent pre- transfer editing is indeed a significant pathway contributing up to 30 % to overall Escherichia coli IleRS editing. Using this methodology in a quantitive analysis of the synthetic site mutants, we found that the conserved Tyr59 is important for both synthetic and editing reactions. This indicates that IleRS catalyzes competing aa-tRNA synthesis and Val-AMP hydrolysis in the overlapping subsites with the synthetic site, providing a rationale for hindered evolution of pre-transfer editing as the major proofreading step. Substitutions of Tyr59 with Thr diminished tRNA-dependent pre-transfer editing providing the first determinant of the synthetic site based pre- transfer editing model. We also observed that IleRS consumes significantly less ATP in proofreading than homologous leucyl- and valyl- tRNA synthetases although it maintains the similar rate of misacylation with these enzymes. As IleRS experiences more error-prone environment in E. coli, relatively unique operational mechanism in the synthetic and editing pathways in IleRS seems to be driven by cellular energetic requirements.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA