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Transfer RNA methyltransferases from Thermoplasma acidophilum, a thermoacidophilic archaeon.

Kawamura T, Anraku R, Hasegawa T, Tomikawa C, Hori H - Int J Mol Sci (2014)

Bottom Line: We found that the Ta0997, Ta0931, and Ta0836 genes of T. acidophilum encode Trm1, Trm56 and Trm5, respectively.However, the speed of methyl-transfer by Trm56 to the precursor RNA was considerably slower than that to the mature transcript, which suggests that Trm56 acts mainly on the transcript after the intron has been removed.Moreover, cellular arrangements of the tRNA methyltransferases in T. acidophilum are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime Univsersity, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan. t.kwmr.0115@gmail.com.

ABSTRACT
We investigated tRNA methyltransferase activities in crude cell extracts from the thermoacidophilic archaeon Thermoplasma acidophilum. We analyzed the modified nucleosides in native initiator and elongator tRNAMet, predicted the candidate genes for the tRNA methyltransferases on the basis of the tRNAMet and tRNALeu sequences, and characterized Trm5, Trm1 and Trm56 by purifying recombinant proteins. We found that the Ta0997, Ta0931, and Ta0836 genes of T. acidophilum encode Trm1, Trm56 and Trm5, respectively. Initiator tRNAMet from T. acidophilum strain HO-62 contained G+, m1I, and m22G, which were not reported previously in this tRNA, and the m2G26 and m22G26 were formed by Trm1. In the case of elongator tRNAMet, our analysis showed that the previously unidentified G modification at position 26 was a mixture of m2G and m22G, and that they were also generated by Trm1. Furthermore, purified Trm1 and Trm56 could methylate the precursor of elongator tRNAMet, which has an intron at the canonical position. However, the speed of methyl-transfer by Trm56 to the precursor RNA was considerably slower than that to the mature transcript, which suggests that Trm56 acts mainly on the transcript after the intron has been removed. Moreover, cellular arrangements of the tRNA methyltransferases in T. acidophilum are discussed.

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T. acidophilum Trm1 methylates the initiator tRNAMet transcript. (A) Given that the 5'-end of the T. acidophilum initiator tRNAMet is A, the transcript is not synthesized efficiently by T7 RNA polymerase. Consequently, the initiator tRNAMet transcript was synthesized with a 5'-leader sequence by T7 RNA polymerase, purified by 10% PAGE (7 M urea), and then the 5'-leader sequence was removed with E. coli RNase P; (B) The cleavage of the 5'-leader sequence was monitored by 10% PAGE (7 M urea). The gel was stained with toluidine blue. The lanes are as follows: left, initiator tRNAMet with the 5'-leader sequence; center, the reaction mixture; right, purified mature initiator tRNAMet transcript. The M1 RNA in RNase P can be observed at the top of the gel in the center lane; (C) The methylated nucleotide was analyzed by 2D-TLC.
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ijms-16-00091-f006: T. acidophilum Trm1 methylates the initiator tRNAMet transcript. (A) Given that the 5'-end of the T. acidophilum initiator tRNAMet is A, the transcript is not synthesized efficiently by T7 RNA polymerase. Consequently, the initiator tRNAMet transcript was synthesized with a 5'-leader sequence by T7 RNA polymerase, purified by 10% PAGE (7 M urea), and then the 5'-leader sequence was removed with E. coli RNase P; (B) The cleavage of the 5'-leader sequence was monitored by 10% PAGE (7 M urea). The gel was stained with toluidine blue. The lanes are as follows: left, initiator tRNAMet with the 5'-leader sequence; center, the reaction mixture; right, purified mature initiator tRNAMet transcript. The M1 RNA in RNase P can be observed at the top of the gel in the center lane; (C) The methylated nucleotide was analyzed by 2D-TLC.

Mentions: 1 During the course of the current study, it has been reported that Sulfolobus acidocaldarius TrmJ generates the Cm32 modification in tRNA. The candidate gene in T. acidophilum was predicted by a BLAST search; 2 The sequence of the initiator tRNAMet that is encoded in the genome of the T. acidophilum strain HO-62 differs from that reported in the earlier study [25] (see Figure 1A and Figure 6A): the nucleotide at position 57 is A instead of G in strain HO-62. Furthermore, the tRNA gene in the genome of strain HO-62 contains additional nucleotides, A20b and C22; 3 The Ta0852 gene product was expressed in Escherichia coli as a soluble protein. However, we could not detect any ability to form m1A58; ?, the corresponding enzyme is unknown.


Transfer RNA methyltransferases from Thermoplasma acidophilum, a thermoacidophilic archaeon.

Kawamura T, Anraku R, Hasegawa T, Tomikawa C, Hori H - Int J Mol Sci (2014)

T. acidophilum Trm1 methylates the initiator tRNAMet transcript. (A) Given that the 5'-end of the T. acidophilum initiator tRNAMet is A, the transcript is not synthesized efficiently by T7 RNA polymerase. Consequently, the initiator tRNAMet transcript was synthesized with a 5'-leader sequence by T7 RNA polymerase, purified by 10% PAGE (7 M urea), and then the 5'-leader sequence was removed with E. coli RNase P; (B) The cleavage of the 5'-leader sequence was monitored by 10% PAGE (7 M urea). The gel was stained with toluidine blue. The lanes are as follows: left, initiator tRNAMet with the 5'-leader sequence; center, the reaction mixture; right, purified mature initiator tRNAMet transcript. The M1 RNA in RNase P can be observed at the top of the gel in the center lane; (C) The methylated nucleotide was analyzed by 2D-TLC.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307237&req=5

ijms-16-00091-f006: T. acidophilum Trm1 methylates the initiator tRNAMet transcript. (A) Given that the 5'-end of the T. acidophilum initiator tRNAMet is A, the transcript is not synthesized efficiently by T7 RNA polymerase. Consequently, the initiator tRNAMet transcript was synthesized with a 5'-leader sequence by T7 RNA polymerase, purified by 10% PAGE (7 M urea), and then the 5'-leader sequence was removed with E. coli RNase P; (B) The cleavage of the 5'-leader sequence was monitored by 10% PAGE (7 M urea). The gel was stained with toluidine blue. The lanes are as follows: left, initiator tRNAMet with the 5'-leader sequence; center, the reaction mixture; right, purified mature initiator tRNAMet transcript. The M1 RNA in RNase P can be observed at the top of the gel in the center lane; (C) The methylated nucleotide was analyzed by 2D-TLC.
Mentions: 1 During the course of the current study, it has been reported that Sulfolobus acidocaldarius TrmJ generates the Cm32 modification in tRNA. The candidate gene in T. acidophilum was predicted by a BLAST search; 2 The sequence of the initiator tRNAMet that is encoded in the genome of the T. acidophilum strain HO-62 differs from that reported in the earlier study [25] (see Figure 1A and Figure 6A): the nucleotide at position 57 is A instead of G in strain HO-62. Furthermore, the tRNA gene in the genome of strain HO-62 contains additional nucleotides, A20b and C22; 3 The Ta0852 gene product was expressed in Escherichia coli as a soluble protein. However, we could not detect any ability to form m1A58; ?, the corresponding enzyme is unknown.

Bottom Line: We found that the Ta0997, Ta0931, and Ta0836 genes of T. acidophilum encode Trm1, Trm56 and Trm5, respectively.However, the speed of methyl-transfer by Trm56 to the precursor RNA was considerably slower than that to the mature transcript, which suggests that Trm56 acts mainly on the transcript after the intron has been removed.Moreover, cellular arrangements of the tRNA methyltransferases in T. acidophilum are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime Univsersity, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan. t.kwmr.0115@gmail.com.

ABSTRACT
We investigated tRNA methyltransferase activities in crude cell extracts from the thermoacidophilic archaeon Thermoplasma acidophilum. We analyzed the modified nucleosides in native initiator and elongator tRNAMet, predicted the candidate genes for the tRNA methyltransferases on the basis of the tRNAMet and tRNALeu sequences, and characterized Trm5, Trm1 and Trm56 by purifying recombinant proteins. We found that the Ta0997, Ta0931, and Ta0836 genes of T. acidophilum encode Trm1, Trm56 and Trm5, respectively. Initiator tRNAMet from T. acidophilum strain HO-62 contained G+, m1I, and m22G, which were not reported previously in this tRNA, and the m2G26 and m22G26 were formed by Trm1. In the case of elongator tRNAMet, our analysis showed that the previously unidentified G modification at position 26 was a mixture of m2G and m22G, and that they were also generated by Trm1. Furthermore, purified Trm1 and Trm56 could methylate the precursor of elongator tRNAMet, which has an intron at the canonical position. However, the speed of methyl-transfer by Trm56 to the precursor RNA was considerably slower than that to the mature transcript, which suggests that Trm56 acts mainly on the transcript after the intron has been removed. Moreover, cellular arrangements of the tRNA methyltransferases in T. acidophilum are discussed.

Show MeSH
Related in: MedlinePlus