In vitro synthesis of selenocysteinyl-tRNA(UCA) from seryl-tRNA(UCA): involvement and characterization of the selD gene product.

Leinfelder, Walfred; Forchhammer, Karl; Veprek, B.; Zehelein, E; Böck, August

doi:10.1073/pnas.87.2.543

Cited by 173 publications

(84 citation statements)

References 30 publications

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“…This method proposed here will also be applicable to direct analysis of the conversion of amino acids attached to tRNAs, e.g. selenocystein tRNA [22] and glutamate tRNA for the formation of O-aminolevulinate [23], and tRNA having mischarging activity [21] in vivo, because two or more amino acids attached to the tRNA would be easily detected on the TLC plate. We expect that this method will be useful for research into tRNA identity in vivo and for the analysis of presently unknown functions of RNAs in vivo.…”

Section: Resultsmentioning

confidence: 99%

A new method for identifying the amino acid attached to a particular RNA in the cell

1996

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To investigate the function of tRNAs or any other aminoacylable RNAs in vivo, it is important to be able to estimate the amounts and species of aminoacylated RNAs in living cells. We have developed a method of analyzing amino acids attached to particular tRNAs obtained from cells. After the ester bond between the amino acid and the 3'-adenosine moiety of a specific aminoacyl-tRNA is stabilized by acetylation of the amino acid with [14C]acetic anhydride, the aminoacyl-tRNA can be fished out with a solid-phase-attached DNA probe. The t4C-labeled acetylamino acid is then released from the thus purified acetyl-aminoacyl-tRNAs by alkaline treatment and detected by TLC analysis.

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Section: Resultsmentioning

confidence: 99%

A new method for identifying the amino acid attached to a particular RNA in the cell

1996

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“…Besides aminoacylation, the assay might be used to measure tRNA deacylation rates, catalyzed by aaRS in a posttransfer editing reaction (35). In addition, the assay can be adapted easily for the assay of other enzymes that use aa-tRNA as a substrate including tRNA fMet formylase (36), tRNA-transamidase (37), and possibly selenocysteine syntase (38). It should be possible also to adapt the assay to measure the rate of peptide-bond formation catalyzed by the ribosome.…”

Section: Discussionmentioning

confidence: 99%

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Wolfson

Uhlenbeck

2002

Proc. Natl. Acad. Sci. U.S.A.

124

163

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A highly sensitive assay of tRNA aminoacylation was developed that directly measures the fraction of aminoacylated tRNA by following amino acid attachment to the 3 -32 P-labeled tRNA. When applied to Escherichia coli alanyl-tRNA synthetase, the assay allowed accurate measurement of aminoacylation of the most deleterious mutants of tRNA Ala . The effect of tRNA Ala identity mutations on both aminoacylation efficiency (kcat͞KM) and steady-state level of aminoacyl-tRNA was evaluated in the absence and presence of inorganic pyrophosphatase and elongation factor Tu. Significant levels of aminoacylation were achieved for tRNA mutants even when the kcat͞KM value is reduced by as much as several thousandfold. These results partially reconcile the discrepancy between in vivo and in vitro analysis of tRNA Ala identity.T wo major approaches have been developed to identify nucleotides required for tRNA recognition by aminoacyl-tRNA (aa-tRNA) synthetases (aaRSs). The first involves introducing a suppressor anticodon into a tRNA of interest and then examining the suppressor activity of the point mutations in vivo (1). The second involves assaying aminoacylation activity of mutant tRNAs made by transcription in vitro (2). Both methods reveal a limited number of nucleotides critical for aminoacylation, the importance of which was confirmed by performing ''swap'' experiments in which the proposed nucleotides were transplanted into the body of another tRNA (2, 3). Subsequent analysis of aminoacylation rate in vitro or sequencing of the reporter proteins in vivo was used to confirm that change in tRNA identity had occurred. These two methods generally agree reasonably well with each other and correlate well with observed base-specific contacts in cocrystal structures of tRNA-aaRS complexes (4-6). However, there are several examples where the conclusions derived from in vitro and in vivo approaches disagree, the most notable of which is the recognition of tRNA Ala by Escherichia coli alanyl-tRNA synthetase (AlaRS). Although no cocrystal structure is available, in vitro experiments with truncated RNA substrates (7, 8) and mutant tRNA Ala (9) as well as identity-swap experiments (10, 11) strongly suggested that the G3⅐U70 pair is the major identity element of tRNA Ala . The deleterious effect of mutations of this base pair significantly exceeds those of other tRNA Ala recognition elements (8, 9, 12-15) and clearly was indicative of the predominant role of this base pair and in particular the exocyclic amino group of G3 in recognition. In contrast, in vivo experiments showed that many of same mutations in the G3⅐U70 base pair have only a moderate effect on the levels of aminoacylated tRNA Ala in E. coli and consequently on the ability of the mutant tRNAs to function in protein synthesis (16,17). A careful analysis of the in vivo experiments indicates that the observed discrepancy was not caused by the technical problems associated with the determining of tRNA aminoacylation levels in vivo, and therefore it was proposed that the discre...

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“…The physiological cofactor(s) of these deiodinase selenoenzymes is (are) unknown. phate (Leinfelder et al, 1990), which is generated from H 2 Se and ATP (Lacourciere, 1999;Lacourciere and Stadtman, 1999).…”

Section: The Biosynthesis Of Selenoproteinsmentioning

confidence: 99%

Selenium in Biology: Facts and Medical Perspectives

Köhrl¹,

Brigelius‐Flohé²,

Böck³

et al. 2000

Biological Chemistry

247

143

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Several decades after the discovery of selenium as an essential trace element in vertebrates approximately 20 eukaryotic and more than 15 prokaryotic selenoproteins containing the 21 st proteinogenic amino acid, selenocysteine, have been identified, partially characterized or cloned from several species. Many of these proteins are involved in redox reactions with selenocysteine acting as an essential component of the catalytic cycle. Enzyme activities have been assigned to the glutathione peroxidase family, to the thioredoxin reductases, which were recently identified as selenoproteins, to the iodothyronine deiodinases, which metabolize thyroid hormones, and to the selenophosphate synthetase 2, which is involved in selenoprotein biosynthesis. Prokaryotic selenoproteins catalyze redox reactions and formation of selenoethers in (stress-induced) metabolism and energy production of E. coli, of the clostridial cluster XI and of other prokaryotes. Apart from the specific and complex biosynthesis of selenocysteine, selenium also reversibly binds to proteins, is incorporated into selenomethionine in bacteria, yeast and higher plants, or posttranslationally modifies a catalytically essential cysteine residue of CO dehydrogenase. Expression of individual eukaryotic selenoproteins exhibits high tissue specificity, depends on selenium availability, in some cases is regulated by hormones, and if impaired contributes to several pathological conditions. Disturbance of selenoprotein expression or function is associated with deficiency syndromes (Keshan and Kashin-Beck disease), might contribute to tumorigenesis and atherosclerosis, is altered in several bacterial and viral infections, and leads to infertility in male rodents.

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In vitro synthesis of selenocysteinyl-tRNA(UCA) from seryl-tRNA(UCA): involvement and characterization of the selD gene product.

Cited by 173 publications

References 30 publications

A new method for identifying the amino acid attached to a particular RNA in the cell

A new method for identifying the amino acid attached to a particular RNA in the cell

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase

Selenium in Biology: Facts and Medical Perspectives

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In vitro synthesis of selenocysteinyl-tRNA(UCA) from seryl-tRNA(UCA): involvement and characterization of the selD gene product.

Cited by 173 publications

References 30 publications

A new method for identifying the amino acid attached to a particular RNA in the cell

A new method for identifying the amino acid attached to a particular RNA in the cell

Modulation of tRNA Ala identity by inorganic pyrophosphatase

Selenium in Biology: Facts and Medical Perspectives

Contact Info

Product

Resources

About

Modulation of tRNA ^Ala identity by inorganic pyrophosphatase