Detection of Enzymatic Activity of Transfer RNA Modification Enzymes Using Radiolabeled tRNA Substrates

Grosjean, Henri; Droogmans, Louis; Roovers, Martine; Keith, Gérard

doi:10.1016/s0076-6879(07)25003-7

Cited by 103 publications

(144 citation statements)

References 112 publications

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“…The sequence coding the last 194 amino acids of TADA that comprises the deaminase domain (DN-TADA) was expressed in E. coli fused to an N-terminal His-tag, and the activity of the purified soluble protein fraction was tested on in vitro-synthesized cp-tRNA Arg (ACG) labeled with [ 32 P]ATP. The presence of inosine was analyzed by thin layer chromatography (TLC) as described (Grosjean et al, 2007). Onedimensional TLC showed a radioactive spot comigrating with inosine monophosphate (IMP), as a consequence of adenosine deaminase activity by DN-TADA ( Figure 5A1, lane c).…”

Section: The Deaminase Domain Of Tada Is Structurally Similar To Othementioning

confidence: 99%

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Arabidopsis tRNA Adenosine Deaminase Arginine Edits the Wobble Nucleotide of Chloroplast tRNAArg(ACG) and Is Essential for Efficient Chloroplast Translation

et al. 2009

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RNA editing changes the coding/decoding information relayed by transcripts via nucleotide insertion, deletion, or conversion. Editing of tRNA anticodons by deamination of adenine to inosine is used both by eukaryotes and prokaryotes to expand the decoding capacity of individual tRNAs. This limits the number of tRNA species required for codon-anticodon recognition. We have identified the Arabidopsis thaliana gene that codes for tRNA adenosine deaminase arginine (TADA), a chloroplast tRNA editing protein specifically required for deamination of chloroplast (cp)-tRNA Arg (ACG) to cp-tRNA Arg (ICG). Land plant TADAs have a C-terminal domain similar in sequence and predicted structure to prokaryotic tRNA deaminases and also have very long N-terminal extensions of unknown origin and function. Biochemical and mutant complementation studies showed that the C-terminal domain is sufficient for cognate tRNA deamination both in vitro and in planta. Disruption of TADA has profound effects on chloroplast translation efficiency, leading to reduced yields of chloroplast-encoded proteins and impaired photosynthetic function. By contrast, chloroplast transcripts accumulate to levels significantly above those of wild-type plants. Nevertheless, absence of cp-tRNA Arg (ICG) is compatible with plant survival, implying that two out of three CGN codon recognition occurs in chloroplasts, though this mechanism is less efficient than wobble pairing.

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Section: The Deaminase Domain Of Tada Is Structurally Similar To Othementioning

confidence: 99%

“…Incubation conditions were as described (Wolf et al, 2002). Afterwards, the tRNA substrates were digested with nuclease P1 to generate 59-monophosphate nucleosides that were analyzed by one-or two-dimensional TLC as described (Grosjean et al, 2007).…”

Section: Trna Deaminase Activity Test In Vitromentioning

confidence: 99%

Arabidopsis tRNA Adenosine Deaminase Arginine Edits the Wobble Nucleotide of Chloroplast tRNAArg(ACG) and Is Essential for Efficient Chloroplast Translation

et al. 2009

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“…Post-transcriptional modifications at the wobble position 34 and the purine 37, 3' adjacent to the anticodon, are known to be involved in stabilizing the geometry of the anticodon loop and its dynamics for optimum codon/anticodon interaction [55,56]. Early work on tRNA analysis used combinations of column, paper, and thin layer chromatography of partial and complete nuclease digests of tRNAs to identify and characterize modified nucleosides [2,3]. Recent analyses include separation of tRNA fragments or nucleoside mixtures by liquid chromatography followed by mass spectrometric analysis of individual peak fractions (LC-MS), pioneered by the laboratories of Nishimura, McCloskey and Crain [57,58].…”

Section: Base Modificationsmentioning

confidence: 99%

“…Analysis of tRNAs, therefore, constitutes an integral part of studies of protein synthesis. Methods for tRNA purification and sequence analysis, including the determination of base modifications, are essential for structure-function studies and have been described elsewhere [2,3]. Methods for separation of tRNAs on polyacrylamide gels under native or denaturing conditions followed by identification of individual tRNAs by Northern hybridization have also proven to be extremely useful.…”

Section: Introductionmentioning

confidence: 99%

The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases

Köhrer

RajBhandary

2008

Methods

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Here we describe the many applications of acid urea polyacrylamide gel electrophoresis (acid urea PAGE) followed by Northern blot analysis to studies of tRNAs. Acid urea PAGE allows the electrophoretic separation of different forms of a tRNA, discriminated by changes in bulk, charge, and/or conformation that are brought about by aminoacylation, formylation, or modification of a tRNA. Among the examples described are (i) analysis of the effect of mutations in the Escherichia coli initiator tRNA on its aminoacylation and formylation; (ii) evidence of orthogonality of suppressor tRNAs in mammalian cells and yeast; (iii) analysis of aminoacylation specificity of an archaeal prolyl-tRNA synthetase that can aminoacylate archaeal tRNA Pro with cysteine, but does not aminoacylate archaeal tRNA Cys with cysteine; (iv) identification and characterization of the AUAdecoding minor tRNA Ile in archaea; and (v) evidence that the archaeal minor tRNA Ile contains a modified base in the wobble position different from lysidine found in the corresponding eubacterial tRNA.

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“…3 The rule for the vast majority of the remaining 100 or so modifications is, that a modified RNA cannot be easily distinguished from its unmodified counterpart, because a small modification such as e.g., a methylation does only minor changes to the RNA's physicochemical properties. Therefore, many methods first break down the RNA chain by nuclease digestion, 4 in some cases followed by a phosphatase treatment (shown in Fig. 2).…”

Section: Identification By Physicochemical Propertiesmentioning

confidence: 99%

Detection of RNA modifications

Kellner

Burhenne²,

Helm³

2010

RNA Biology

120

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Detection of Enzymatic Activity of Transfer RNA Modification Enzymes Using Radiolabeled tRNA Substrates

Cited by 103 publications

References 112 publications

Arabidopsis tRNA Adenosine Deaminase Arginine Edits the Wobble Nucleotide of Chloroplast tRNAArg(ACG) and Is Essential for Efficient Chloroplast Translation

Arabidopsis tRNA Adenosine Deaminase Arginine Edits the Wobble Nucleotide of Chloroplast tRNAArg(ACG) and Is Essential for Efficient Chloroplast Translation

The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases

Detection of RNA modifications

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