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.
Highlights
Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at 59 °C and pH 1.9 [1]
T. acidophilum has been investigated from various viewpoints as described above, there is little knowledge about tRNA modifications, with the exception of some early studies [24,25,26,27]
The candidate gene in T. acidophilum was predicted by a BLAST search; 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]: the nucleotide at position 57 is A
Summary
Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at 59 °C and pH 1.9 [1]. The energy metabolism of T. acidophilum has been studied in detail because it can grow under an extreme microaerophilic environment [17,18]. T. acidophilum has been investigated from various viewpoints as described above, there is little knowledge about tRNA modifications, with the exception of some early studies [24,25,26,27]. We found that the m7G modification was present at a novel position, nucleotide 49 in class II tRNALeu (Figure 1C); class II tRNAs have a long variable region. We found several distinct modifications in this tRNALeu (Figure 1C): 4-thiouridine at position 9. We tested the tRNA methyltransferase activities in crude cell extract from T. acidophilum, analyzed the methylated nucleosides in purified tRNAs and characterized the tRNA methyltransferases by expressing recombinant proteins in Escherichia coli
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