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

Abstract

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.

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

“…2E). In our previous study, this transcript was well methylated by A. aeolicus TrmB [17]. Thus, this result demonstrates the importance of the D-arm structure in RNA recognition by Trm8-Trm82.…”

“…2), because threedimensional structure of yeast tRNA Phe was established [6,7]. The methyl acceptance activities of these truncated transcripts by A. aeolicus tRNA (m 7 G46) methyltransferase were previously investigated: the transcripts F, G, and H in Fig 2 were well methylated and transcripts C, E and I were slowly methylated [17]. After the annealing, we measured CD-spectra of the transcripts at various temperatures (15,20,25,30,35, 40, 45, 50, 55, 60, 65 and 70°C) to check the conformation.…”

“…Recently, it has been reported that yeast tRNA (m 7 G46) methyltransferase is composed of two protein subunits (Trm8 and Trm82) and their genes have been identified [15]. More recently, bacterial genes have also been identified as trmB (classical name, yggH) from E. coli [16] and Aquifex aeolicus [17]. A gene disruption mutant of yeast [15] or E. coli [16] has revealed that the tRNA (m 7 G46) methyltransferase activity is not essential for cell viability.…”

“…The A. aeolicus enzyme has an extra C-terminal portion and a shortened N-terminus as compared to its mesophilic counterpart from E. coli [16] or B. subtilis [21]. Our previous study revealed that the most important recognition sites of the A. aeolicus enzyme are included in the T-arm structure of tRNA [17], suggesting that A. aeolicus TrmB accesses the G46 residue from the T-stem side. This eubacterial enzyme is composed of only a TrmB subunit [17].…”

“…Our previous study revealed that the most important recognition sites of the A. aeolicus enzyme are included in the T-arm structure of tRNA [17], suggesting that A. aeolicus TrmB accesses the G46 residue from the T-stem side. This eubacterial enzyme is composed of only a TrmB subunit [17]. In contrast, the yeast enzyme is a heterodimer composed of two protein subunits (Trm8 and Trm82) [15].…”