A convenient procedure for preparing transfer ribonucleic acid from Escherichia coli

Avital, Shlomo; Elson, David

doi:10.1016/0005-2787(69)90038-0

Cited by 80 publications

(21 citation statements)

References 19 publications

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“…One of these missing species eluted at 26 min (fraction 52) and the other at 32 min (fraction 64). Two candidate modified nucleosides that would be expected to elute at these positions are mcm 5 5 U eluted exactly with the 26-min peak missing in the F6 deletion mutant and mcm 5 s 2 U eluted exactly with the 32-min peak also absent in the F6 deletion mutant (data not shown). We thus designate the F6 (YML014w) gene TRM9 (for tRNA methyltransferase).…”

Section: Resultsmentioning

confidence: 90%

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Novel Methyltransferase for Modified Uridine Residues at the Wobble Position of tRNA

Kalhor

Clarke

2003

Molecular and Cellular Biology

148

172

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We have identified a novel tRNA methyltransferase in Saccharomyces cerevisiae that we designate Trm9. This enzyme, the product of theYML014w gene, catalyzes the esterification of modified uridine nucleotides, resulting in the formation of 5-methylcarbonylmethyluridine in tRNA Arg3 and 5-methylcarbonylmethyl-2-thiouridine in tRNA Glu . In intact yeast cells, disruption of the TRM9 gene results in the complete loss of these modified wobble bases and increased sensitivity at 37°C to paromomycin, a translational inhibitor. These results suggest a role for this potentially reversible methyl esterification reaction when cells are under stress.In the last few years, it has become increasingly evident that S-adenosylmethionine (AdoMet)-dependent methyltransferases play a wide range of roles in cell physiology. Unlike other covalent modifications such as phosphorylation that occur mainly on proteins, methylation has been observed in DNA, RNA, proteins, lipids, polysaccharides, and small molecules (15,35,36,38). As a result of such diversity, identifying the methyltransferases and their substrates has been challenging. Although more than 25 methylated nucleotides have been documented for eukaryotic tRNAs alone, only a few of the corresponding enzymes and their genes have been identified (16,47).In 1999, using conserved AdoMet binding motifs, we identified 26 putative methyltransferases (designated F1 to F26) encoded by the genome of the yeast Saccharomyces cerevisiae (40). Biochemically, we have been able to show that two of these species are in fact methyltransferases; F3 (YDR465c) catalyzes the transfer of a methyl group to the delta-nitrogen atom of arginine residues in a novel protein posttranslational reaction (40, 58), while F9 (YER175c) catalyzes the methyl esterification of the small molecule trans-aconitate (9). Recently, F1 (YDL201w) was found to be responsible for formation of 7-methylguanosine at position 46 of tRNA (3).One major approach in identifying novel enzymes has been the comparison of the methylation spectra of strains lacking the putative methyltransferase and those of their isogenic wildtype parents by separation techniques such as sodium dodecyl sulfate (SDS) gel electrophoresis (26, 59). In the yeast S. cerevisiae, one can study biological methylation in vivo by incubating cells with, the major biological methyl donor (26). As a result, all methyl-accepting species such as RNAs, proteins, and small molecules can become radiolabeled and the fate of the methylated species can be followed biochemically. One can then look for differences in the methylation spectra between a mutant strain and its parent.In all domains of life, tRNAs are highly modified posttranscriptionally (46, 47). Methylation reactions account for the majority of these modifications. In the yeast S. cerevisiae, eight tRNA methyltransferases have been identified so far. The TRM1 gene product forms N 2 ,N 2 -dimethylguanosine at position 26 (14). TRM2 encodes a protein that forms 5-methyluridine at position 54 (41). TRM3 encodes a pr...

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Section: Resultsmentioning

confidence: 90%

“…The aqueous phase was collected and mixed with 2.5 volumes of ethanol and 0.1 volume of 20% potassium acetate to precipitate RNA. tRNA was purified with the 2 M LiCl extraction method as described previously (5). The tRNA pellet was washed twice with 80% ethanol and finally dissolved in water or 2.4 M tetraethylammonium chloride.…”

Section: Methodsmentioning

confidence: 99%

Novel Methyltransferase for Modified Uridine Residues at the Wobble Position of tRNA

Kalhor

Clarke

2003

Molecular and Cellular Biology

148

172

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“…Total tRNA for HPLC analysis was prepared essentially as described (Björk et al 2001) followed by LiCl fractionation (Avital and Elson 1969). For HPLC analysis 50 µg tRNA was digested to nucleosides by using nuclease P1 and bacterial alkaline phosphatase (Gehrke et al 1982), and the hydrolysate was analyzed by HPLC (Gehrke and Kuo 1990).…”

Section: Rna Methodsmentioning

confidence: 99%

The Saccharomyces cerevisiae TAN1 gene is required for N⁴-acetylcytidine formation in tRNA

Johansson¹,

Byström²

2004

RNA

102

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“…Total RNA was prepared by using TRIzol reagent (GIBCO͞BRL) from a frozen and homogenized L. forficatus centipede collected in Ann Arbor, MI. To eliminate most of the rRNA, we modified previously published protocols (8,9) as follows. Total RNA was dissolved in 200 l of 2 M LiCl͞0.1 M potassium acetate, pH 5.0.…”

Section: Methodsmentioning

confidence: 99%

A novel type of RNA editing occurs in the mitochondrial tRNAs of the centipede Lithobius forficatus

Lavrov

Brown

Boore

2000

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

295

204

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We determined the complete mtDNA sequence of the centipede Lithobius forficatus and found that only one of the 22 inferred tRNA genes encodes a fully paired aminoacyl acceptor stem. The other 21 genes encode tRNAs with up to five mismatches in these stems, and some of these overlap extensively with the downstream genes. Because a well-paired acceptor stem is required for proper tRNA functioning, RNA editing in the products of these genes was suspected. We investigated this hypothesis by studying cDNA sequences from eight tRNAs and found the editing of up to 5 nt at their 3 ends. This editing appears to occur by a novel mechanism with the 5 end of the acceptor stem being used as a template for the de novo synthesis of the 3 end, presumably by an RNA-dependent RNA polymerase. In addition, unusual secondary structures for several tRNAs were found, including those lacking a T⌿C (T) or a dihydrouridine (D) arm, and having an unusual number of base pairs in the acceptor or anticodon stems.

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A convenient procedure for preparing transfer ribonucleic acid from Escherichia coli

Cited by 80 publications

References 19 publications

Novel Methyltransferase for Modified Uridine Residues at the Wobble Position of tRNA

Novel Methyltransferase for Modified Uridine Residues at the Wobble Position of tRNA

The Saccharomyces cerevisiae TAN1 gene is required for N⁴-acetylcytidine formation in tRNA

A novel type of RNA editing occurs in the mitochondrial tRNAs of the centipede Lithobius forficatus

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A convenient procedure for preparing transfer ribonucleic acid from Escherichia coli

Cited by 80 publications

References 19 publications

Novel Methyltransferase for Modified Uridine Residues at the Wobble Position of tRNA

Novel Methyltransferase for Modified Uridine Residues at the Wobble Position of tRNA

The Saccharomyces cerevisiae TAN1 gene is required for N4-acetylcytidine formation in tRNA

A novel type of RNA editing occurs in the mitochondrial tRNAs of the centipede Lithobius forficatus

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The Saccharomyces cerevisiae TAN1 gene is required for N⁴-acetylcytidine formation in tRNA