The physiological target for LeuRS translational quality control is norvaline

Abstract

The fidelity of protein synthesis depends on the capacity of aminoacyl-tRNA synthetases (AARSs) to couple only cognate amino acidtRNA pairs. If amino acid selectivity is compromised, fidelity can be ensured by an inherent AARS editing activity that hydrolyses mischarged tRNAs. Here, we show that the editing activity of Escherichia coli leucyl-tRNA synthetase (EcLeuRS) is not required to prevent incorrect isoleucine incorporation. Rather, as shown by kinetic, structural and in vivo approaches, the prime biological function of LeuRS editing is to prevent mis-incorporation of the non-standard amino acid norvaline. This conclusion follows from a reassessment of the discriminatory power of LeuRS against isoleucine and the demonstration that a LeuRS editing-deficient E. coli strain grows normally in high concentrations of isoleucine but not under oxygen deprivation conditions when norvaline accumulates to substantial levels. Thus, AARS-based translational quality control is a key feature for bacterial adaptive response to oxygen deprivation. The non-essential role for editing under normal bacterial growth has important implications for the development of resistance to antimicrobial agents targeting the LeuRS editing site.

Homologous trans -editing factors with broad tRNA specificity prevent mistranslation caused by serine/threonine misactivation

“…Cellular degradation and apoptosis caused by a mutation in the editing domain of ValRS have been reported in murine cells (16). In addition, editing defects in bacteria often result in slower growth rates, delayed growth, or even death (17)(18)(19)(20)(21)(22).…”

Degenerate Connective Polypeptide 1 (CP1) Domain from Human Mitochondrial Leucyl-tRNA Synthetase

“…hmtLeuRS Only Has tRNA-independent Pre-transfer Editing for Nva-Recent studies showed that Met 40 in EcLeuRS plays a key part in discriminating Ile; prevention of incorrect Nva incorporation is the major biological editing activity of EcLeuRS (21). Our study determined systematically the catalytic efficiency of hmtLeuRS toward different analogs of Leu.…”

“…They indicated that hmtLeuRS could discriminate Ile efficiently via a precise synthetic core (18,20). However, it was suggested recently that the main targets of LeuRS editing include Nva (21). Thus, whether hmtLeuRS is able to rigorously discriminate Nva and other Leu analogs (such as ABA) during aminoacylation needs to be further explored.…”

Multiple Quality Control Pathways Limit Non-protein Amino Acid Use by Yeast Cytoplasmic Phenylalanyl-tRNA Synthetase

“…Aliquots of the supernatant (typically 4 l) were injected onto a reverse phase HPLC column (Phenomenex Kinetex C18, 2.1 ϫ 150 mm, 1.7-m particle size, 100 Å pore size) equilibrated in solvent A and isocratically eluted (100 l/m). The effluent from the column was directly connected to an electrospray ion source (Agilent Jet Stream) attached to a triple quadrupole mass spectrometer (Agilent 6460) scanning in the positive multiple reaction monitoring mode with standard resolution settings (FWHM 0.7) using previously optimized conditions for the following transitions: L-DOPA, 198 Liquid Chromatographic Purification of Amino Acids-The accuracy of kinetic analyses of non-cognate amino acid substrates for enzyme specificity determination depends on the degree of cognate amino acid contamination (14). Therefore, we purified p-Tyr, DL-o-Tyr, and L-DOPA before their use for kinetic analyses.…”