scholarly journals Adenosine triphosphate consumption by bacterial arginyl-transfer ribonucleic acid synthetases

1979 ◽  
Vol 179 (2) ◽  
pp. 407-412 ◽  
Author(s):  
J M Godeau ◽  
J Charlier
Keyword(s):  
Escherichia Coli ◽  
Adenosine Triphosphate ◽  
Ribonucleic Acid ◽  
Adenosine Triphosphatase ◽  
Bacillus Stearothermophilus ◽  
Trna Synthetase ◽  
Luciferase Assay ◽  
Trna Synthetases ◽  
E Coli ◽  
Firefly Luciferin

ATP consumption by arginyl-tRNA synthetases from Escherichia coli and Bacillus stearothermophilus has been investigated by the firefly luciferin–luciferase assay. Arginyl-tRNA synthetase from E. coli utilizes ATP only for aminocylation of tRNA with a 1:1 stoicheiometry. In contrast, we have shown an adenosine triphosphatase activity of arginyl-tRNA synthetase from B. stearothermophilus in the absence of tRNAArg. Dowex chromatography revealed the formation of ADP by the thermophile enzyme; under aminoacylation conditions, AMP was also formed in amounts stoicheiometric with arginyl-tRNA formation.

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 A comparison of purified valyl-transfer ribonucleic acid synthetase from Bacillus stearothermophilus and from Escherichia coli

1974 ◽  
Vol 139 (2) ◽  
pp. 391-398 ◽  
Author(s):  
Susan Wilkinson ◽  
Jeremy R. Knowles
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Bacillus Stearothermophilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Binding Constants ◽  
Trna Synthetase ◽  
Subunit Structure ◽  
Filtration Method ◽  
E Coli

The purification of valyl-tRNA synthetase from Bacillus stearothermophilus is described. The protein was greater than 90% homogeneous on polyacrylamide-gel electrophoresis after more than 850-fold purification. It has a molecular weight of 110000, and no evidence was found for the presence of subunit structure. The properties of the purified enzyme were compared with those of purified valyl-tRNA synthetase from Escherichia coli. The thermal stability, pH-stability and dependence of activity on the temperature and pH of the assay are reported. The two enzymes recognize and charge tRNAVal from crude tRNA of the mesophile E. coli and of the thermophile B. stearothermophilus, indiscriminately. The gel-filtration method was extended to measure the binding of tRNA to synthetase directly. Binding constants for tRNAVal to valyl-tRNA synthetase from B. stearothermophilus were determined between 5° and 60°C.

scholarly journals Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand

1977 ◽  
Vol 167 (2) ◽  
pp. 419-428 ◽  
Author(s):  
C M Clarke ◽  
J R Knowles
Keyword(s):  
Ribonucleic Acid ◽  
Bacillus Stearothermophilus ◽  
Preceding Paper ◽  
Trna Synthetase ◽  
Rapid Preparation ◽  
Purification Method ◽  
Trna Synthetases ◽  
Trna Species ◽  
Aminoacyl Transfer ◽  
Cognate Trna

The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417].

scholarly journals Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Biomolecules ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 255 ◽  
Author(s):  
Sviatlana Smolskaya ◽  
Yaroslav Andreev
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Nonsense Suppression ◽  
Expression Vectors ◽  
Site Specific ◽  
Suppressor Trna ◽  
E Coli ◽  
Orthogonal Pair

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

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