[22] Experimental proof for the misactivation of amino acids by aminoacyl-tRNA synthetases

Gl, Igloi; F, von der Haar; Cramer, Friedrich

doi:10.1016/0076-6879(79)59091-0

Cited by 29 publications

(19 citation statements)

References 10 publications

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“…DMAPP-tRNA Transferase Assay. DMAPP-tRNA transferase activity was assayed by a modified TCA-precipitation procedure (Igloi et al, 1979). tRNA Phe was heat annealed just prior to use by placing samples in a 75 °C water bath which was allowed to cool gradually to 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Escherichia coli Dimethylallyl Diphosphate:tRNA Dimethylallyltransferase: A Binding Mechanism for Recombinant Enzyme

Poulter

1997

Biochemistry

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Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the first step in the biosynthesis of the hypermodified A37 residue in tRNAs that read codons beginning with uridine. The enzyme, encoded by the miaA gene, was overproduced and purified to apparent homogeneity in three steps by ion-exchange (DE52 and Mono-Q) and size exclusion chromatography. Affinity-tagged DMAPP-tRNA transferase containing a C-terminal tripeptide alpha-tubulin epitope also was overproduced and purified to apparent homogeneity in two steps by ion-exchange and immunoaffinity chromatography. Addition of the C-terminal tripeptide alpha-tubulin epitope to DMAPP-tRNA transferase did not affect the activity of the enzyme. Undermodified tRNA(Phe) used as substrate in the DMAPP-tRNA transferase-catalyzed reaction was isolated and purified from an overexpressing clone in a miaA deficient strain of E. coli. Active recombinant E. coli DMAPP-tRNA transferase is monomeric. The enzyme transferred the dimethylallyl moiety of DMAPP to A37, located adjacent to the anticodon in undermodified tRNA(Phe). The enzyme required Mg2+ for activity and exhibited a broad pH optimum. Michaelis constants for tRNA(Phe) and DMAPP are 96 +/- 11 nM and 3.2 +/- 0.5 microM, respectively, and Vmax = 0.83 +/- 0.02 micromol min-1 mg-1. DMAPP-tRNA transferase bound tRNA(Phe) with a dissociation constant of 5.2 +/- 1.2 nM. In contrast, DMAPP did not bind to the enzyme in the absence of tRNA. However, DMAPP was bound with a dissociation constant of 3.4 +/- 0.6 microM in the presence of a minihelix analogue of the anticodon stem-loop of tRNA(Phe) where the base corresponding to A37 was replaced by inosine. These results suggest an ordered sequential mechanism for substrate binding.

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Section: Methodsmentioning

confidence: 99%

Escherichia coli Dimethylallyl Diphosphate:tRNA Dimethylallyltransferase: A Binding Mechanism for Recombinant Enzyme

Poulter

1997

Biochemistry

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“…The reaction was stopped after 10 min by adding 100 µL of ice-cold 10% TCA. Precipitation was done via a modified TCA precipitation protocol [ 65 ]. The whole volume of reaction tube was transferred to a Whatman filter paper and air dried for 15 min.…”

Section: Methodsmentioning

confidence: 99%

The Effect of tRNA[Ser]Sec Isopentenylation on Selenoprotein Expression

Fradejas‐Villar

Bohleber

Zhao

et al. 2021

IJMS

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Transfer RNA[Ser]Sec carries multiple post-transcriptional modifications. The A37G mutation in tRNA[Ser]Sec abrogates isopentenylation of base 37 and has a profound effect on selenoprotein expression in mice. Patients with a homozygous pathogenic p.R323Q variant in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder, and hence we wondered whether selenoprotein expression was impaired. Patient fibroblasts with the homozygous p.R323Q variant did not show a general decrease in selenoprotein expression. However, recombinant human TRIT1R323Q had significantly diminished activities towards several tRNA substrates in vitro. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Mass-spectrometry revealed that hypermodification of U34 to mcm5Um occurs independently of isopentenylation of A37 in tRNA[Ser]Sec. Western blotting and 75Se metabolic labeling showed only moderate effects on selenoprotein levels and 75Se incorporation. A detailed analysis of Trit1-deficient liver using ribosomal profiling demonstrated that UGA/Sec re-coding was moderately affected in Selenop, Txnrd1, and Sephs2, but not in Gpx1. 2′O-methylation of U34 in tRNA[Ser]Sec depends on FTSJ1, but does not affect UGA/Sec re-coding in selenoprotein translation. Taken together, our results show that a lack of isopentenylation of tRNA[Ser]Sec affects UGA/Sec read-through but differs from a A37G mutation.

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“…Aliquots of 10 µ were taken after 2,4,6,8,10,20,30, 40, and 60 min and spotted onto charcoal filter disks. The disks were washed twice for 10 min in 1.5% trichloracetic acid containing 40 mM unlabeled pyrophosphate and once for 5 min in water and dried and the radioactivity was counted ( Simlot & Pfaender, 1973;Igloi et al, 1978Igloi et al, , 1979. AMP Formation.…”

Section: Methodsmentioning

confidence: 99%

“…Aminoacylation assays contained (in a total volume of 0.1 mL) 300 µ [14C]amino acid, 10 mM ATP, and 1 mg/mL unfractionated tRNA from yeast or E. coli in 150 mM Tris/HCl, pH 8.0, containing 150 mM KC1, 10 mM MgS04, and 5 mM /3-mercaptoethanol. The reaction was started by addition of 5 µg of valyl-tRNA synthetase (Igloi et al, 1978(Igloi et al, , 1979Jakubowski & Fersht, 1981) and incubated at 37 °C. After 2, 5, and 10 min, 10-µ aliquots were withdrawn and spotted onto 3-mm filter disks.…”

Section: Methodsmentioning

confidence: 99%

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Fidelity in the aminoacylation of tRNAVal with hydroxy analogs of valine, leucine, and isoleucine by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli

Englisch-Peters¹,

Haar²,

Cramer³

1990

Biochemistry

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Several analogues of valine, leucine, and isoleucine carrying hydroxyl groups in the gamma- or delta-position have been tested in the aminoacylation of tRNA by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli. Results of the ATP/PPi exchange and of the aminoacylation reactions indicate that the amino acid analogues not only can form the aminoacyl adenylate intermediate but are also transferred to tRNA. However, the fact that the reaction consumes an excess of ATP indicates that the misactivated amino acid analogue is hydrolytically removed. Thus, valyl-tRNA synthetase from S. cerevisiae shows a high fidelity in forming valyl-tRNA. Although the much bulkier amino acid analogues allo- and iso-gamma-hydroxyvaline and allo- and iso-gamma-hydroxyisoleucine are initially charged to tRNA, the misaminoacylated tRNA(Val) is enzymatically deacylated. This cleavage reaction is mediated by the hydroxyl groups of the amino acid analogues which are converted into the corresponding lactones.

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[22] Experimental proof for the misactivation of amino acids by aminoacyl-tRNA synthetases

Cited by 29 publications

References 10 publications

Escherichia coli Dimethylallyl Diphosphate:tRNA Dimethylallyltransferase: A Binding Mechanism for Recombinant Enzyme

Escherichia coli Dimethylallyl Diphosphate:tRNA Dimethylallyltransferase: A Binding Mechanism for Recombinant Enzyme

The Effect of tRNA[Ser]Sec Isopentenylation on Selenoprotein Expression

Fidelity in the aminoacylation of tRNAVal with hydroxy analogs of valine, leucine, and isoleucine by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli

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