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

One integral step in the transition from a nucleic acid encoded-genome to functional proteins is the aminoacylation of tRNA molecules. To perform this activity, aminoacyl-tRNA synthetases (aaRSs) activate free amino acids in the cell forming an aminoacyl-adenylate before transferring the amino acid on to its cognate tRNA. These newly formed aminoacyl-tRNA (aa-tRNA) can then be used by the ribosome during mRNA decoding. In Escherichia coli, there are twenty aaRSs encoded in the genome, each of which corresponds to one of the twenty proteinogenic amino acids used in translation. Given the shared chemicophysical properties of many amino acids, aaRSs have evolved mechanisms to prevent erroneous aa-tRNA formation with non-cognate amino acid substrates. Of particular interest is the post-transfer proofreading activity of alanyl-tRNA synthetase (AlaRS) which prevents the accumulation of Ser-tRNAAla and Gly-tRNAAla in the cell. We have previously shown that defects in AlaRS proofreading of Ser-tRNAAla lead to global dysregulation of the E. coli proteome, subsequently causing defects in growth, motility, and antibiotic sensitivity. Here we report second-site AlaRS suppressor mutations that alleviate the aforementioned phenotypes, revealing previously uncharacterized residues within the AlaRS proofreading domain that function in quality control.

Download Full-text

Relaxed Substrate Specificity Leads to Extensive tRNA Mischarging by Streptococcus pneumoniae Class I and Class II Aminoacyl-tRNA Synthetases

mBio ◽

10.1128/mbio.01656-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 8

Author(s):

Jennifer Shepherd ◽

Michael Ibba

Keyword(s):

Quality Control ◽

Cell Wall ◽

Protein Synthesis ◽

Streptococcus Pneumoniae ◽

Amino Acid ◽

Substrate Specificity ◽

Trna Synthetase ◽

Content Type ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases

ABSTRACTAminoacyl-tRNA synthetases provide the first step in protein synthesis quality control by discriminating cognate from noncognate amino acid and tRNA substrates. While substrate specificity is enhanced in many instances bycis-andtrans-editing pathways, it has been revealed that in organisms such asStreptococcus pneumoniaesome aminoacyl-tRNA synthetases display significant tRNA mischarging activity. To investigate the extent of tRNA mischarging in this pathogen, the aminoacylation profiles of class I isoleucyl-tRNA synthetase (IleRS) and class II lysyl-tRNA synthetase (LysRS) were determined. Pneumococcal IleRS mischarged tRNAIlewith both Val, as demonstrated in other bacteria, and Leu in a tRNA sequence-dependent manner. IleRS substrate specificity was achieved in an editing-independent manner, indicating that tRNA mischarging would only be significant under growth conditions where Ile is depleted. Pneumococcal LysRS was found to misaminoacylate tRNALyswith Ala and to a lesser extent Thr and Ser, with mischarging efficiency modulated by the presence of an unusual U4:G69 wobble pair in the acceptor stems of both pneumococcal tRNALysisoacceptors. Addition of thetrans-editing factor MurM, which also functions in peptidoglycan synthesis, reduced Ala-tRNALysproduction by LysRS, providing evidence for cross talk between the protein synthesis and cell wall biogenesis pathways. Mischarging of tRNALysby AlaRS was also observed, and this would provide additional potential MurM substrates. More broadly, the extensive mischarging activities now described for a number ofStreptococcus pneumoniaeaminoacyl-tRNA synthetases suggest that adaptive misaminoacylation may contribute significantly to the viability of this pathogen during amino acid starvation.IMPORTANCEStreptococcus pneumoniaeis a common causative agent of several debilitating and potentially life-threatening infections, such as pneumonia, meningitis, and infectious endocarditis. Such infections are increasingly difficult to treat due to widespread development of penicillin resistance. High-level penicillin resistance is known to depend in part upon MurM, a protein involved in both aminoacyl-tRNA-dependent synthesis of indirect amino acid cross-linkages within cell wall peptidoglycan and in translation quality control. The involvement of MurM in both protein synthesis and antibiotic resistance identify it as a potential target for the development of new and potent antibiotics for pneumococcal infections. The goals of this work were to identify and characterizeS. pneumoniaepathways that can synthesize mischarged tRNAs and to relate these activities to expected changes in protein and peptidoglycan biosynthesis during antibiotic and nutritional stress.

Download Full-text

The First Proteomic Study of Nostoc sp. PCC 7120 Exposed to Cyanotoxin BMAA under Nitrogen Starvation

Toxins ◽

10.3390/toxins12050310 ◽

2020 ◽

Vol 12 (5) ◽

pp. 310 ◽

Cited By ~ 3

Author(s):

Olga A. Koksharova ◽

Ivan O. Butenko ◽

Olga V. Pobeguts ◽

Nina A. Safronova ◽

Vadim M. Govorun

Keyword(s):

Amino Acid ◽

Nitrogen Metabolism ◽

Nitrogen Starvation ◽

Practical Interest ◽

Trna Synthetase ◽

Water Soluble ◽

Protein Amino Acid ◽

Heterocyst Differentiation ◽

Proteomic Study ◽

Pcc 7120

The oldest prokaryotic photoautotrophic organisms, cyanobacteria, produce many different metabolites. Among them is the water-soluble neurotoxic non-protein amino acid beta-N-methylamino-L-alanine (BMAA), whose biological functions in cyanobacterial metabolism are of fundamental scientific and practical interest. An early BMAA inhibitory effect on nitrogen fixation and heterocyst differentiation was shown in strains of diazotrophic cyanobacteria Nostoc sp. PCC 7120, Nostoc punctiforme PCC 73102 (ATCC 29133), and Nostoc sp. strain 8963 under conditions of nitrogen starvation. Herein, we present a comprehensive proteomic study of Nostoc (also called Anabaena) sp. PCC 7120 in the heterocyst formation stage affecting by BMAA treatment under nitrogen starvation conditions. BMAA disturbs proteins involved in nitrogen and carbon metabolic pathways, which are tightly co-regulated in cyanobacteria cells. The presented evidence shows that exogenous BMAA affects a key nitrogen regulatory protein, PII (GlnB), and some of its protein partners, as well as glutamyl-tRNA synthetase gltX and other proteins that are involved in protein synthesis, heterocyst differentiation, and nitrogen metabolism. By taking into account the important regulatory role of PII, it becomes clear that BMAA has a severe negative impact on the carbon and nitrogen metabolism of starving Nostoc sp. PCC 7120 cells. BMAA disturbs carbon fixation and the carbon dioxide concentrating mechanism, photosynthesis, and amino acid metabolism. Stress response proteins and DNA repair enzymes are upregulated in the presence of BMAA, clearly indicating severe intracellular stress. This is the first proteomic study of the effects of BMAA on diazotrophic starving cyanobacteria cells, allowing a deeper insight into the regulation of the intracellular metabolism of cyanobacteria by this non-protein amino acid.

Download Full-text

Orphan designation: 505 amino acid protein, corresponding to amino acids 2-506 of the wild-type human histidyl-tRNA synthetase, Treatment of limb-girdle muscular dystrophy

Case Medical Research ◽

10.31525/cmr-2935ce2 ◽

2020 ◽

Author(s):

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Muscular Dystrophy ◽

Trna Synthetase ◽

Limb Girdle Muscular Dystrophy ◽

Wild Type ◽

Orphan Designation ◽

Acid Protein ◽

Amino Acid Protein

Download Full-text

Nucleotide and deduced amino acid sequence of human threonyl-tRNA synthetase reveals extensive homology to the Escherichia coli and yeast enzymes

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)92906-6 ◽

1991 ◽

Vol 266 (15) ◽

pp. 9919-9923

Author(s):

M.E. Cruzen ◽

S.M. Arfin

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Trna Synthetase

Download Full-text

Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽

10.1007/s00726-020-02927-z ◽

2020 ◽

Author(s):

Thomas L. Williams ◽

Debra J. Iskandar ◽

Alexander R. Nödling ◽

Yurong Tan ◽

Louis Y. P. Luk ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Genetic Code Expansion ◽

Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

Download Full-text