Naslov
Synthetic and editing reactions of aminoacyl-tRNA synthetases using cognate and non-cognate amino acid substrates

Autori
Cvetešić, Nevena ; Gruić-Sovulj, Ita

Izvornik
Methods (1046-2023) 113 (2016); 13-26

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
Aminoacyl-tRNA synthetases ; Proofreading ; tRNA-dependent pre-transfer editing ; Post-transfer editing ; Single-turnover kinetics ; Non-cognate amino acids

Sažetak
The covalent coupling of cognate amino acid-tRNA pairs by corresponding aminoacyl-tRNA synthetases (aaRS) defines the genetic code and provides aminoacylated tRNAs for ribosomal protein synthesis. Besides the cognate substrate, some non-cognate amino acids may also compete for tRNA aminoacylation. However, their participation in protein synthesis is generally prevented by an aaRS proofreading activity located in the synthetic site and in a separate editing domain. These mechanisms, coupled with the ability of certain aaRSs to discriminate well against non-cognate amino acids in the synthetic reaction alone, define the accuracy of the aminoacylation reaction. aaRS quality control may also act as a gatekeeper for the standard genetic code and prevents infiltration by natural amino acids that are not normally coded for protein biosynthesis. This latter finding has reinforced interest in understanding the principles that govern discrimination against a range of potential non-cognate amino acids. This paper presents an overview of the kinetic assays that have been established for monitoring synthetic and editing reactions with cognate and non-cognate amino acid substrates. Taking into account the peculiarities of non-cognate reactions, the specific controls needed and the dedicated experimental designs are discussed in detail. Kinetic partitioning within the synthetic and editing sites controls the balance between editing and aminoacylation. We describe in detail steady-state and single-turnover approaches for the analysis of synthetic and editing reactions, which ultimately enable mechanisms of amino acid discrimination to be determined.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA