A chimaeric glutamyl:glutaminyl-tRNA synthetase: implications for evolution

2008 ◽  
Vol 417 (2) ◽  
pp. 449-455 ◽  
Author(s):  
Rajesh Saha ◽  
Saumya Dasgupta ◽  
Gautam Basu ◽  
Siddhartha Roy
Keyword(s):  
Catalytic Domain ◽  
Trna Synthetase ◽  
Binding Domain ◽  
Temperature Sensitive ◽  
Trna Synthetases ◽  
E Coli ◽  
Discrimination Capability ◽  
Binding Domains ◽  
History Of ◽  
Substrate Binding Domain

aaRSs (aminoacyl-tRNA synthetases) are multi-domain proteins that have evolved by domain acquisition. The anti-codon binding domain was added to the more ancient catalytic domain during aaRS evolution. Unlike in eukaryotes, the anti-codon binding domains of GluRS (glutamyl-tRNA synthetase) and GlnRS (glutaminyl-tRNA synthetase) in bacteria are structurally distinct. This originates from the unique evolutionary history of GlnRSs. Starting from the catalytic domain, eukaryotic GluRS evolved by acquiring the archaea/eukaryote-specific anti-codon binding domain after branching away from the eubacteria family. Subsequently, eukaryotic GlnRS evolved from GluRS by gene duplication and horizontally transferred to bacteria. In order to study the properties of the putative ancestral GluRS in eukaryotes, formed immediately after acquiring the anti-codon binding domain, we have designed and constructed a chimaeric protein, cGluGlnRS, consisting of the catalytic domain, Ec GluRS (Escherichia coli GluRS), and the anti-codon binding domain of EcGlnRS (E. coli GlnRS). In contrast to the isolated EcN-GluRS, cGluGlnRS showed detectable activity of glutamylation of E. coli tRNAglu and was capable of complementing an E. coli ts (temperature-sensitive)-GluRS strain at non-permissive temperatures. Both cGluGlnRS and EcN-GluRS were found to bind E. coli tRNAglu with native EcGluRS-like affinity, suggesting that the anticodon-binding domain in cGluGlnRS enhances kcat for glutamylation. This was further confirmed from similar experiments with a chimaera between EcN-GluRS and the substrate-binding domain of EcDnaK (E. coli DnaK). We also show that an extended loop, present in the anticodon-binding domains of GlnRSs, is absent in archaeal GluRS, suggesting that the loop was a later addition, generating additional anti-codon discrimination capability in GlnRS as it evolved from GluRS in eukaryotes.

Molecular cloning and regulation of expression of the genes for initiation factor 3 and two aminoacyl-tRNA synthetases

1982 ◽  
Vol 152 (1) ◽  
pp. 357-362
Author(s):  
D Elseviers ◽  
P Gallagher ◽  
A Hoffman ◽  
B Weinberg ◽  
I Schwartz
Keyword(s):  
Trna Synthetase ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Translation System ◽  
Temperature Sensitive ◽  
Regulation Of Expression ◽  
Trna Synthetases ◽  
E Coli ◽  
Translation Initiation Factor 3

A 22-kilobase fragment of the Escherichia coli chromosome which contains the genes for translation initiation factor 3, phenylalanyl-tRNA synthetase, and threonyl-tRNA synthetase was cloned into plasmid pACYC184. The hybrid plasmid (designated pID1) complements a temperature-sensitive pheS lesion in E. coli NP37. pID1-transformed NP37 overproduce initiation factor 3 and phenylalanyl-tRNA synthetase. Gene expression from pID1 was studied in vitro in a coupled transcription-translation system and in minicells. The results suggest that the genes for initiation factor 3 and phenylalanyl- and threonyl-tRNA synthetase are regulated by different mechanisms.

Glutamyl and Glutaminyl-tRNA Synthetases Are a Promising Target for the Design of Threonine-Producing Strain

2019 ◽  
Vol 35 (6) ◽  
pp. 39-50
Author(s):  
T.V. Yuzbashev ◽  
A.S. Fedorov ◽  
F.V. Bondarenko ◽  
A.S. Savchenko ◽  
T.V. Vybornaya ◽  
...  
Keyword(s):  
Biomass Accumulation ◽  
Trna Synthetase ◽  
Temperature Sensitive ◽  
Original Strain ◽  
Trna Synthetases ◽  
Resource Center ◽  
Promising Target ◽  
The Russian Federation ◽  
Increase In Productivity ◽  
Culture Growth

The present work describes an approach that improves the properties of the strain producing L-threonine via the reduction in the biomass accumulation during fermentation. Glutamyl- and glutaminyl-tRNA synthetases were chosen as targets. Mutants carrying temperature-sensitive alleles were obtained. It was shown that the used system caused the suppression of the function of tRNA synthetases which led to a rapid arrest of the culture growth, and an increase in productivity and yield of the L-threonine synthesis. One of the temperature-sensitive strains was used to obtain under non-permissive conditions of mutants with the suppressed above phenotype. Some of these mutants accumulate less biomass and produce by 10-12% more threonine than the original strain. Escherichia coli, producing strain, threonine, aminoacyl-tRNA synthetase, ts-mutation This work was supported by the Ministry of Science and Higher Education of the Russian Federation (project code RFMEFI61017X0011), and it was carried out using the equipment of the National Bio-Resource Center All-Russian Collection of Industrial Microorganisms, NRC «Kurchatov Institute» - GosNIIgenetika.

scholarly journals Inhibitory mechanism of reveromycin A at the tRNA binding site of a class I synthetase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bingyi Chen ◽  
Siting Luo ◽  
Songxuan Zhang ◽  
Yingchen Ju ◽  
Qiong Gu ◽  
...  
Keyword(s):  
Binding Site ◽  
Catalytic Domain ◽  
Trna Synthetase ◽  
Rational Drug Design ◽  
Class I ◽  
Binding Assays ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Rational Drug ◽  
Trna Binding

AbstractThe polyketide natural product reveromycin A (RM-A) exhibits antifungal, anticancer, anti-bone metastasis, anti-periodontitis and anti-osteoporosis activities by selectively inhibiting eukaryotic cytoplasmic isoleucyl-tRNA synthetase (IleRS). Herein, a co-crystal structure suggests that the RM-A molecule occupies the substrate tRNAIle binding site of Saccharomyces cerevisiae IleRS (ScIleRS), by partially mimicking the binding of tRNAIle. RM-A binding is facilitated by the copurified intermediate product isoleucyl-adenylate (Ile-AMP). The binding assays confirm that RM-A competes with tRNAIle while binding synergistically with l-isoleucine or intermediate analogue Ile-AMS to the aminoacylation pocket of ScIleRS. This study highlights that the vast tRNA binding site of the Rossmann-fold catalytic domain of class I aminoacyl-tRNA synthetases could be targeted by a small molecule. This finding will inform future rational drug design.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli

Genes ◽  
2018 ◽  
Vol 9 (10) ◽  
pp. 473 ◽  
Author(s):  
Takuya Umehara ◽  
Saori Kosono ◽  
Dieter Söll ◽  
Koji Tamura
Keyword(s):  
Escherichia Coli ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Lysine Acetylation ◽  
Trna Synthetases ◽  
E Coli ◽  
Domains Of Life ◽  
The Impact

Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which Nε-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis.

scholarly journals Amber suppression in mammalian cells dependent upon expression of an Escherichia coli aminoacyl-tRNA synthetase gene.

1996 ◽  
Vol 16 (3) ◽  
pp. 907-913 ◽  
Author(s):  
H J Drabkin ◽  
H J Park ◽  
U L RajBhandary
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Trna Gene ◽  
Trna Synthetase ◽  
Developmentally Regulated ◽  
Coding Sequence ◽  
Suppressor Trna ◽  
Trna Synthetases ◽  
E Coli ◽  
Nonsense Codons

As an approach to inducible suppression of nonsense mutations in mammalian and in higher eukaryotic cells, we have analyzed the expression of an Escherichia coli glutamine-inserting amber suppressor tRNA gene in COS-1 and CV-1 monkey kidney cells. The tRNA gene used has the suppressor tRNA coding sequence flanked by sequences derived from a human initiator methionine tRNA gene and has two changes in the coding sequence. This tRNA gene is transcribed, and the transcript is processed to yield the mature tRNA in COS-1 and CV-1 cells. We show that the tRNA is not aminoacylated in COS-1 cells by any of the endogenous aminoacyl-tRNA synthetases and is therefore not functional as a suppressor. Concomitant expression of the E. coli glutaminyl-tRNA synthetase gene results in aminoacylation of the suppressor tRNA and its functioning as a suppressor. These results open up the possibility of attempts at regulated suppression of nonsense codons in mammalian cells by regulating expression of the E. coli glutaminyl-tRNA synthetase gene in an inducible, cell-type specific, or developmentally regulated manner.

scholarly journals High-throughput aminoacyl-tRNA synthetase engineering for genetic code expansion in yeast

2021 ◽  
Author(s):  
Jessica T. Stieglitz ◽  
James A. Van Deventer
Keyword(s):  
Genetic Code ◽  
High Throughput ◽  
Trna Synthetase ◽  
Noncanonical Amino Acids ◽  
Trna Synthetases ◽  
E Coli ◽  
Biological Studies ◽  
Screening Strategies ◽  
Genetic Code Expansion ◽  
Translation Systems

Protein expression with genetically encoded noncanonical amino acids (ncAAs) benefits a broad range of applications, from the discovery of biological therapeutics to fundamental biological studies. A major factor limiting the use of ncAAs is the lack of orthogonal translation systems (OTSs) that support efficient genetic code expansion at repurposed stop codons. Aminoacyl-tRNA synthetases (aaRSs) have been extensively evolved in E. coli but are not always orthogonal in eukaryotes. In this work, we use a yeast display-based ncAA incorporation reporter platform with fluorescence-activated cell sorting (FACS) to screen libraries of aaRSs in high throughput for 1) incorporation of ncAAs not previously encoded in yeast; 2) improvement of the performance of an existing aaRS; 3) highly selective OTSs capable of discriminating between closely related ncAA analogs; and 4) OTSs exhibiting enhanced polyspecificity to support translation with structurally diverse sets of ncAAs. The number of previously undiscovered aaRS variants we report in this work more than doubles the total number of translationally active aaRSs available for genetic code manipulation in yeast. The success of myriad screening strategies has important implications related to the fundamental properties and evolvability of aaRSs. Furthermore, access to OTSs with diverse activities and specific/polyspecific properties are invaluable for a range of applications within chemical biology, synthetic biology, and protein engineering.

scholarly journals Construction and Analysis of HybridEscherichia coli-Bacillus subtilis dnaK Genes

1999 ◽  
Vol 181 (6) ◽  
pp. 1971-1974 ◽  
Author(s):  
Axel Mogk ◽  
Bernd Bukau ◽  
Rolf Lutz ◽  
Wolfgang Schumann
Keyword(s):  
Bacillus Subtilis ◽  
Functional Unit ◽  
Substrate Binding ◽  
Binding Domain ◽  
Atpase Domain ◽  
E Coli ◽  
Terminal Domain ◽  
Species Specific ◽  
Dnak Protein ◽  
Substrate Binding Domain

ABSTRACT The highly conserved DnaK chaperones consist of an N-terminal ATPase domain, a central substrate-binding domain, and a C-terminal domain whose function is not known. Since Bacillus subtilis dnaK was not able to complement an Escherichia coli dnaK null mutant, we performed domain element swap experiments to identify the regions responsible for this finding. It turned out that the B. subtilis DnaK protein needed approximately normal amounts of the cochaperone DnaJ to be functional in E. coli. The ATPase domain and the substrate-binding domain form a species-specific functional unit, while the C-terminal domains, although less conserved, are exchangeable. Deletion of the C-terminal domain in E. coli DnaK affected neither complementation of growth at high temperatures nor propagation of phage λ but abolished degradation of ς32.

scholarly journals Lysyl-tRNA Synthetase-generated Lysyl-Adenylate Is a Substrate for Histidine Triad Nucleotide Binding Proteins

2006 ◽  
Vol 282 (7) ◽  
pp. 4719-4727 ◽  
Author(s):  
Tsui-Fen Chou ◽  
Carston R. Wagner
Keyword(s):  
Binding Proteins ◽  
Nucleotide Binding ◽  
Trna Synthetase ◽  
Site Directed Mutagenesis ◽  
Trna Synthetases ◽  
E Coli ◽  
Protein Protein Interaction ◽  
Nucleotide Binding Proteins ◽  
Micromolar Range

Histidine triad nucleotide binding proteins (Hints) are the most ancient members of the histidine triad protein superfamily of nucleotidyltransferases and hydrolyases. Protein-protein interaction studies have found that complexes of the transcription factors MITF or USF2 and lysyl-tRNA synthetase (LysRS) are associated with human Hint1. Therefore, we hypothesized that lysyl-AMP or the LysRS·lysyl-AMP may be a native substrate for Hints. To explore the biochemical relationship between Hint1 and LysRS, a series of catalytic radiolabeling, mutagenesis, and kinetic experiments was conducted with purified LysRSs and Hints from human and Escherichia coli. After incubation of the E. coli or human LysRS with Hints and [α-32P]ATP, but not [α-32P]GTP, 32P-labeled Hints were observed. By varying time and the concentrations of lysine, Mg2+, or LysRS, the adenylation of Hint was found to be dependent on the formation of lysyl-AMP. Site-directed mutagenesis studies of the active site histidine triad revealed that Hint labeling could be abolished by substitution of either His-101 of E. coli hinT or His-112 of human Hint1 by either alanine or glycine. Ap4A, believed to be synthesized by LysRS in vivo, and Zn2+ were shown to inhibit the formation of Hint-AMP with an IC50 value in the low micromolar range. Consistent with pyrophosphate being an inhibitor for aminoacyl-tRNA synthetase, incubations in the presence of pyrophosphatase resulted in enhanced formation of Hint-AMP. These results demonstrate that the lysyl-AMP intermediate formed by LysRS is a natural substrate for Hints and suggests a potential highly conserved regulatory role for Hints on LysRS and possibly other aminoacyl-tRNA synthetases.

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