scholarly journals Engineered Aminoacyl-tRNA Synthetase for Cell-Selective Analysis of Mammalian Protein Synthesis

2016 ◽  
Vol 138 (13) ◽  
pp. 4278-4281 ◽  
Author(s):  
Alborz Mahdavi ◽  
Graham D. Hamblin ◽  
Granton A. Jindal ◽  
John D. Bagert ◽  
Cathy Dong ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Selective Analysis ◽  
Mammalian Protein

scholarly journals Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase v1

2020 ◽  
Author(s):  
Anne Zemella ◽  
Theresa Richter ◽  
Lena Thoring ◽  
Stefan Kubick
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Trna Synthetase ◽  
Fluorescent Labeling ◽  
Laser Scanning Microscopy ◽  
Aminoacyl Trna Synthetase ◽  
Free Protein ◽  
Canonical Amino Acid ◽  
Cell Free Protein Synthesis

This is part 3.1 of the "A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties" collection of protocols: https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven Collection Abstract: Fluorescent labeling of de novo synthesized proteins is in particular a valuable tool for functional and structural studies of membrane proteins. In this context, we present two methods for the site-specific fluorescent labeling of difficult-to-express membrane proteins in combination with cell-free protein synthesis. The cell-free protein synthesis system is based on Chinese Hamster Ovary Cells (CHO) since this system contains endogenous membrane structures derived from the endoplasmic reticulum. These so-called microsomes enable a direct integration of membrane proteins into a biological membrane. In this protocol the first part describes the fluorescent labeling by using a precharged tRNA, loaded with a fluorescent amino acid. The second part describes the preparation of a modified aminoacyl-tRNA-synthetase and a suppressor tRNA that are applied to the CHO cell-free system to enable the incorporation of a non-canonical amino acid. The reactive group of the non-canonical amino acid is further coupled to a fluorescent dye. Both methods utilize the amber stop codon suppression technology. The successful fluorescent labeling of the model G protein-coupled receptor adenosine A2A (Adora2a) is analyzed by in-gel-fluorescence, a reporter protein assay, and confocal laser scanning microscopy (CLSM). Moreover, a ligand-dependent conformational change of the fluorescently labeled Adora2a was analyzed by bioluminescence resonance energy transfer (BRET). For Introduction and Notes, please see: https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven/guidelines

scholarly journals An Archaeal Aminoacyl-tRNA Synthetase Missing from Genomic Analysis

1999 ◽  
Vol 181 (18) ◽  
pp. 5880-5884 ◽  
Author(s):  
Christian S. Hamann ◽  
Kevin R. Sowers ◽  
Richard S. A. Lipman ◽  
Ya-Ming Hou
Keyword(s):  
Protein Synthesis ◽  
Enzyme Activity ◽  
Genomic Analysis ◽  
Trna Synthetase ◽  
Genomic Sequencing ◽  
Aminoacyl Trna Synthetase ◽  
Trna Synthetases ◽  
Cell Lysate ◽  
Aminoacyl Trna Synthetases ◽  
Complete Genomic

ABSTRACT The complete genomic sequencing of Methanococcus jannaschii cannot identify the gene for the cysteine-specific member of aminoacyl-tRNA synthetases. However, we show here that enzyme activity is present in the cell lysate of M. jannaschii. The demonstration of this activity suggests a direct pathway for the synthesis of cysteinyl-tRNACys during protein synthesis.

scholarly journals Regulation of ex-translational activities is the primary function of the multi-tRNA synthetase complex

2020 ◽  
Author(s):  
Haissi Cui ◽  
Mridu Kapur ◽  
Jolene K Diedrich ◽  
John R Yates III ◽  
Susan L Ackerman ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Mrna Translation ◽  
Trna Synthetase ◽  
Stress Conditions ◽  
Aminoacyl Trna Synthetase ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Alternative Function ◽  
Ribosome Pausing

Abstract During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has been proposed to increase protein synthesis efficiency by passing charged tRNAs to ribosomes. An alternative function is that the MSC repurposes specific synthetases that are released from the MSC upon cues for functions independent of translation. To explore this, we generated mammalian cells in which arginyl-tRNA synthetase and/or glutaminyl-tRNA synthetase were absent from the MSC. Protein synthesis, under a variety of stress conditions, was unchanged. Most strikingly, levels of charged tRNAArg and tRNAGln remained unchanged and no ribosome pausing was observed at codons for arginine and glutamine. Thus, increasing or regulating protein synthesis efficiency is not dependent on arginyl-tRNA synthetase and glutaminyl-tRNA synthetase in the MSC. Alternatively, and consistent with previously reported ex-translational roles requiring changes in synthetase cellular localizations, our manipulations of the MSC visibly changed localization.

scholarly journals Fission Yeast Asc1 Stabilizes the Interaction between Eukaryotic Initiation Factor 3a and Rps0A/uS2 for Protein Synthesis

2019 ◽  
Vol 39 (19) ◽  
Author(s):  
Yi-Ting Wang ◽  
Yu-Chen Chien ◽  
Wan-Yi Hsiao ◽  
Chien-Chia Wang ◽  
Shao-Win Wang
Keyword(s):  
Protein Synthesis ◽  
Complex Formation ◽  
Fission Yeast ◽  
Ribosomal Subunit ◽  
Trna Synthetase ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Aminoacyl Trna Synthetase ◽  
Preinitiation Complex ◽  
40S Ribosomal Subunit

ABSTRACT Aminoacyl-tRNA synthetase cofactors play important roles in coordinating aminoacylation and translation. In this study, we describe an additional function of the fission yeast aminoacyl-tRNA synthetase cofactor 1 (Asc1) in translation. We found that Asc1 directly binds and stabilizes the interaction between small ribosomal protein Rps0A/uS2 and eukaryotic initiation factor 3a (eIF3a). In the absence of Asc1, the interaction between eIF3a and Rps0A/uS2 was compromised. The interaction between Rps0A/uS2 and eIF3a mediated the 40S ribosomal subunit binding of eIF3 in 43S preinitiation complex formation to stimulate translation initiation. Keeping with this idea, in an asc1 mutant, the association of mRNA with the 40S ribosomal subunit was defective and protein synthesis was compromised. To show that Asc1 is directly involved in translation, we demonstrate that the addition of recombinant Asc1 is able to rescue the translation defect of the asc1 mutant in a cell-free system. Furthermore, this function of Asc1 is likely to be evolutionarily conserved, as a similar interaction with eIF3a and Rps0A/uS2 could be identified in the budding yeast Saccharomyces cerevisiae and human aminoacyl-tRNA synthetase cofactors. Together, these results identify a function of aminoacyl-tRNA synthetase cofactors in translation preinitiation complex formation, which adds significantly to the expanded functions associated with aminoacyl-tRNA synthetases and their cofactors.

scholarly journals The mechanism of β-N-methylamino-l-alanine inhibition of tRNA aminoacylation and its impact on misincorporation

2019 ◽  
Vol 295 (5) ◽  
pp. 1402-1410 ◽  
Author(s):  
Nien-Ching Han ◽  
Tammy J. Bullwinkle ◽  
Kaeli F. Loeb ◽  
Kym F. Faull ◽  
Kyle Mohler ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Direct Evidence ◽  
Trna Synthetase ◽  
Acid Activation ◽  
Aminoacyl Trna Synthetase ◽  
Amino Acid Activation ◽  
Biochemical Assays ◽  
Serine Codons ◽  
Lateral Sclerosis

β-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS–purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.

scholarly journals Mitochondrial Protein Synthesis and mtDNA Levels Coordinated through an Aminoacyl-tRNA Synthetase Subunit

Cell Reports ◽  
2019 ◽  
Vol 27 (1) ◽  
pp. 40-47.e5 ◽  
Author(s):  
Daria Picchioni ◽  
Albert Antolin-Fontes ◽  
Noelia Camacho ◽  
Claus Schmitz ◽  
Alba Pons-Pons ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mitochondrial Protein ◽  
Trna Synthetase ◽  
Mitochondrial Protein Synthesis ◽  
Aminoacyl Trna Synthetase
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