Aquifex aeolicus tRNA (N2,N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA

Abstract

In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNA Cys has an m 2 2 G26m 2 G27 or m 2 2 G26m 2 2 G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m 2 G26 formation is faster than the m 2 G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.

The open reading frame TTC1157 of Thermus thermophilus HB27 encodes the methyltransferase forming N2-methylguanosine at position 6 in tRNA

“…Sequencing data revealed the presence of m 2 2 G26 and m 2 G/m 2 2 G27 in tRNA Cys of the hyperthermophilic bacterium A. aeolicus. A single MTase of the Trm1 family is responsible for these modifications (Awai et al 2009). Intriguingly, m 2 G at position 6 has been found in only one bacterial tRNA: tRNA Phe of the extreme thermophilic bacterium T. thermophilus.…”

“…The Pf Trm1 enzyme needs specific base-pairing in the D-loop, limited size of the variable loop, and a correct overall tRNA folding for N 2 ,N 2 -dimethylation of G26 (Constantinesco et al 1999). Although initially thought as being excluded from Bacteria, a Trm1 ortholog has been found in the hyperthermophilic bacterium Aquifex aeolicus (Takeda et al 2002;Awai et al 2009). Besides formation of m 2 2 G26, the bacterial Trm1 can also catalyze m 2 G27 formation.…”

“…Besides formation of m 2 2 G26, the bacterial Trm1 can also catalyze m 2 G27 formation. The enzyme transfers the methyl group of S-adenosylmethionine (AdoMet) only in the presence of an intact T-loop (Awai et al 2009). Further, unlike the archaeal Trm1, its bacterial ortholog requires neither specific base-pairing in the D-loop nor a restricted size of the variable loop (Awai et al 2005).…”

Substrate tRNA Recognition Mechanism of a Multisite-specific tRNA Methyltransferase, Aquifex aeolicus Trm1, Based on the X-ray Crystal Structure

“…Moreover, S. cerevisiae Trm7 protein catalyzes 2Ј-O-methylation of nucleotides at positions 32 and 34 (25). As described above, we previously reported that A. aeolicus Trm1 has a multisite specificity (21). Although these examples have been reported, there is little knowledge concerning the mechanism of multisite recognition.…”

“…The responsible gene was first determined to be trm1 from Saccharomyces cerevisiae (13,14) and then experimentally identified from various eukaryotes (Schizosaccharomyces pombe (15), Caenorhabditis elegans (16), and human (17)) and archaea (Pyrococcus furiosus (18), Pyrococcus horikoshii (19), and Haloferax volcanii (20)), consistent with the distribution of the m 2 2 G26 (or m 2 G26) modification in tRNA. Furthermore, we have recently reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a Trm1 protein that catalyzes methyl transfer not only to G26 but also to G27 (21).…”