7-Methylguanine specific tRNA-methyltransferase from Escherichia coli

Aschhoff, H. J.; Elten, H.; Arnold, H.H.; Mahal, G.; Kersten, W.; Kersten, H.

doi:10.1093/nar/3.11.3109

Cited by 31 publications

(7 citation statements)

References 24 publications

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“…A possible explanation for the absence of tRNA (m 7 G46) MTase activity in the GM18 strain would be that trmB encodes a factor influencing yggH expression. Alternatively, two tRNA (m 7 G46) MTases might exist in E. coli, as has been suggested previously (4). However, the fact that the inactivation of the yggH gene leads to a complete absence of tRNA (m 7 G46) MTase activity does not support this hypothesis.…”

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confidence: 81%

The yggH Gene of Escherichia coli Encodes a tRNA (m ⁷ G46) Methyltransferase

Bie

Roovers

Oudjama³

et al. 2003

J Bacteriol

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We cloned, expressed, and purified the Escherichia coli YggH protein and show that it catalyzes the S-adenosyl-L-methionine-dependent formation of N 7 -methylguanosine at position 46 (m 7 G46) in tRNA. Additionally, we generated an E. coli strain with a disrupted yggH gene and show that the mutant strain lacks tRNA (m 7 G46) methyltransferase activity.About 30 different modified nucleosides have been identified in Escherichia coli tRNA. Methylation is one of the most common modifications, and several mutants affected in tRNA methylation have been obtained (5). However, only a few E. coli tRNA methyltransferase (MTase) genes have been cloned and characterized: trmA, trmD, and trmH are involved in the formations of m 5 U54, m 1 G37, and Gm18, respectively (8, 15, 16). Several tRNA MTases have been purified, and the corresponding genes have been mapped on the E. coli chromosome (5, 13), but it has not been convincingly shown which open reading frame (ORF) encodes a given enzyme. On the other hand, evolutionary relationships among various RNA MTase families have been studied and predictions of novel specificities for uncharacterized ORFs have been made (3). Nevertheless, there are still missing links between many known enzymatic activities and predicted RNA MTase genes.As part of a large-scale project aimed at the identification and classification of novel RNA MTases among the uncharacterized or putative proteins in sequence databases, we analyzed the product of the E. coli yggH ORF. This protein exhibits similarity to S-adenosyl-L-methionine (AdoMet)-dependent MTases in the predicted cofactor-binding region but shares no specific amino acid signatures with other families of RNA MTases in the predicted catalytic region, suggesting that it may encode an RNA MTase with a novel specificity. Thus, we selected it for experimental characterization.Amplification and cloning of the yggH ORF. The yggH ORF was PCR amplified from E. coli genomic DNA (strain XL1-Blue) by using Pfu DNA polymerase (Promega). The primers (Table 1) were designed to amplify the yggH ORF with its ribosome binding site. Primers LDB1 and LDB3 were used for the production of a recombinant YggH protein bearing a Cterminal His tag (YggHH6). Primers LDB1 and LDB2 were used for the production of the untagged YggH.The PCR products were cloned into the pCR-BluntII-

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confidence: 81%

The yggH Gene of Escherichia coli Encodes a tRNA (m ⁷ G46) Methyltransferase

Bie

Roovers

Oudjama³

et al. 2003

J Bacteriol

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“…33] [5]. This enzyme activity was first detected in a cell extract of Escherichia coli [8] and has been purified more than 1000 fold [9]. The enzyme activity has also been purified from Salmonella typhimurium [10] and Thermus flavus [11].…”

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confidence: 99%

RNA recognition mechanism of eukaryote tRNA (m⁷G46) methyltransferase (Trm8–Trm82 complex)

et al. 2007

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Yeast tRNA (m 7 G46) methyltransferase contains two protein subunits (Trm8 and Trm82). To address the RNA recognition mechanism of the Trm8-Trm82 complex, we investigated methyl acceptance activities of eight truncated yeast tRNA Phe transcripts. Both the D-stem and T-stem structures were required for efficient methyl-transfer. To clarify the role of the D-stem structure, we tested four mutant transcripts, in which tertiary base pairs were disrupted. The tertiary base pairs were important but not essential for the methyl-transfer to yeast tRNA Phe transcript, suggesting that these base pairs support the induced fit of the G46 base into the catalytic pocket.

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“…The m 7 G46 modification is catalyzed by tRNA (m 7 G46) methyltransferase (tRNA (guanine-N 7 -)-methyltransferase, EC 2.1.1.33). This enzymatic activity was first detected in a cell extract of Escherichia coli (39) and then purified more than 1000-fold (40). The enzyme activity has also been purified from Salmonella typhimurium (41) and Thermus flavus (42).…”

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confidence: 99%

Substrate tRNA Recognition Mechanism of tRNA (m7G46) Methyltransferase from Aquifex aeolicus

Okamoto

Watanabe

Ikeuchi

et al. 2004

Journal of Biological Chemistry

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Transfer RNA (m 7 G46) methyltransferase catalyzes the methyl transfer from S-adenosylmethionine to N 7 atom of the guanine 46 residue in tRNA. Analysis of the Aquifex aeolicus genome revealed one candidate open reading frame, aq065, encoding this gene. The aq065 protein was expressed in Escherichia coli and purified to homogeneity on 15% SDS-polyacrylamide gel electrophoresis. Although the overall amino acid sequence of the aq065 protein differs considerably from that of E. coli YggH, the purified aq065 protein possessed a tRNA (m 7 G46) methyltransferase activity. The modified nucleoside and its location were determined by liquid chromatography-mass spectroscopy. To clarify the RNA recognition mechanism of the enzyme, we investigated the methyl transfer activity to 28 variants of yeast tRNAPhe and E. coli tRNA Thr . It was confirmed that 5-leader and 3-trailer RNAs of tRNA precursor are not required for the methyl transfer. We found that the enzyme specificity was critically dependent on the size of the variable loop. Experiments using truncated variants showed that the variable loop sequence inserted between two stems is recognized as a substrate, and the most important recognition site is contained within the T stem. These results indicate that the Lshaped tRNA structure is not required for methyl acceptance activity. It was also found that nucleotide substitutions around G46 in three-dimensional core decrease the activity.

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7-Methylguanine specific tRNA-methyltransferase from Escherichia coli

Cited by 31 publications

References 24 publications

The yggH Gene of Escherichia coli Encodes a tRNA (m ⁷ G46) Methyltransferase

The yggH Gene of Escherichia coli Encodes a tRNA (m ⁷ G46) Methyltransferase

RNA recognition mechanism of eukaryote tRNA (m⁷G46) methyltransferase (Trm8–Trm82 complex)

Substrate tRNA Recognition Mechanism of tRNA (m7G46) Methyltransferase from Aquifex aeolicus

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7-Methylguanine specific tRNA-methyltransferase from Escherichia coli

Cited by 31 publications

References 24 publications

The yggH Gene of Escherichia coli Encodes a tRNA (m 7 G46) Methyltransferase

The yggH Gene of Escherichia coli Encodes a tRNA (m 7 G46) Methyltransferase

RNA recognition mechanism of eukaryote tRNA (m7G46) methyltransferase (Trm8–Trm82 complex)

Substrate tRNA Recognition Mechanism of tRNA (m7G46) Methyltransferase from Aquifex aeolicus

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The yggH Gene of Escherichia coli Encodes a tRNA (m ⁷ G46) Methyltransferase

The yggH Gene of Escherichia coli Encodes a tRNA (m ⁷ G46) Methyltransferase

RNA recognition mechanism of eukaryote tRNA (m⁷G46) methyltransferase (Trm8–Trm82 complex)