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

Abstract

N 2 -methylguanosine (m 2 G) is found at position 6 in the acceptor stem of Thermus thermophilus tRNA Phe . In this article, we describe the cloning, expression, and characterization of the T. thermophilus HB27 methyltransferase (MTase) encoded by the TTC1157 open reading frame that catalyzes the formation of this modified nucleoside. S-adenosyl-L-methionine is used as donor of the methyl group. The enzyme behaves as a monomer in solution. It contains an N-terminal THUMP domain predicted to bind RNA and contains a C-terminal Rossmann-fold methyltransferase (RFM) domain predicted to be responsible for catalysis. We propose to rename the TTC1157 gene trmN and the corresponding protein TrmN, according to the bacterial nomenclature of tRNA methyltransferases. Inactivation of the trmN gene in the T. thermophilus HB27 chromosome led to a total absence of m 2 G in tRNA but did not affect cell growth or the formation of other modified nucleosides in tRNA Phe . Archaeal homologs of TrmN were identified and characterized. These proteins catalyze the same reaction as TrmN from T. thermophilus. Individual THUMP and RFM domains of PF1002 from Pyrococcus furiosus were produced. These separate domains were inactive and did not bind tRNA, reinforcing the idea that the THUMP domain acts in concert with the catalytic domain to target a particular position of the tRNA molecule.

Long and branched polyamines are required for maintenance of the ribosome, tRNAHis and tRNATyr in Thermus thermophilus cells at high temperatures

“…However, as described previously (Roovers et al . ; Yamagami et al . ), because the in vitro methylation by TrmN causes the second methylation (m 2 2 G6 formation via m 2 G6), the m 2 G6 modification levels were not measured correctly.…”

“…) and TrmN (Roovers et al . ) (Fig. A and B), which catalyze the formation of m 5 U54, m 7 G46, Gm18, m 1 A58 and m 2 G6, respectively, in tRNA Phe (Fig.…”

“…In the case of the m 2 G6 modification by TrmN, the tRNA fraction from the wild-type strain exhibited stronger methyl group acceptance activity than that from the DspeB and DspeD1 strains, suggesting that the m 2 G6 modification level of samples from the DspeB and DspeD1 cells was higher than that from the wild-type cells. However, as described previously (Roovers et al 2012;Yamagami et al 2016), because the in vitro methylation by TrmN causes the second methylation (m 2 2 G6 formation via m 2 G6), the m 2 G6 modification levels were not measured correctly. Thus, although a slight increase in m 2 G6 modification in the DspeB and DspeD1 strains was observed, there was no significant difference in the modification levels of common modified nucleosides in tRNA.…”

“…(J€ uhling et al 2009). Among them, m 2 G was found only at position 6 in tRNA Phe (Grawunder et al 1992;Tomikawa et al 2010;Roovers et al 2012). Thus, m 2 G is a minor modified nucleoside in T. thermophilus tRNA.…”

“…The nucleoside analysis showed that there was no significant difference among the samples from the wildtype, DspeB and DspeD1 strains for the major modified nucleosides. To verify the levels of modified nucleosides in detail, we purified five tRNA methyltransferases, TrmA (Ny & Bj€ ork 1980), TrmB (De Bie et al 2003;Tomikawa et al 2008), TrmH (Hori et al 2002), TrmI (Takuma et al 2015) and TrmN (Roovers et al 2012) (Fig. 8A and B), which catalyze the formation of m 5 U54, m 7 G46, Gm18, m 1 A58 and m 2 G6, respectively, in tRNA Phe (Fig.…”