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.

FIGURE 1.

Inactivation of the TTC1157 gene of T. thermophilus results in the loss of m²G in tRNA. Total tRNA from the T. thermophilus wild-type strain (A) and mutant strain (B) was extracted and hydrolyzed by nuclease P1 and alkaline phosphatase and then analyzed by HPLC. The peak corresponding to m²G is indicated by an arrow. A standard curve with the reference nucleosides C, U, G, A, and m²G is shown in C.

FIGURE 2.

Insertion of a Km^R cassette into the TTC1157 gene of T. thermophilus results in the loss of m²G tRNA-MTase activity. Autoradiography of two-dimensional chromatograms of P1 hydrolysates of [α-³²P] GTP–labeled T. thermophilus tRNA^Phe transcripts incubated for 30 min at 60°C in the presence of AdoMet and 140 μg of crude extract of T. thermophilus wild-type strain (WT) and mutant strain (TTCρKm^R). The number of moles of modified nucleotides per mole of tRNA is indicated between brackets.

FIGURE 3.

Affinity-purified TTC1157 catalyzes the formation of m²G in tRNA^Phe in vitro. (A) SDS-PAGE analysis of the purified TTC1157 protein. (Lane 1) Purified protein; (lane 2) molecular weight (in kiloDaltons) marker (Fermentas). (B) Autoradiography of two-dimensional chromatograms of P1 hydrolysates of [α-³²P] GTP–labeled T. thermophilus tRNA^Phe transcripts incubated for 30 min at 60°C in the presence of purified TTC1157 (for details, see Materials and Methods). Circles of dotted lines show the migration of the pA, pC, and pU nucleotides used as ultraviolet markers. (C) As for B but with one-dimensional chromatography and varying amounts of enzyme. The number of moles of modified nucleotides per mole of tRNA is indicated between brackets.

FIGURE 4.

Formation of m²₂G in tRNA^Phe of T. thermophilus in the presence of high amounts of enzyme. Autoradiography of one-dimensional chromatograms of P1 hydrolysates of [α-³²P] GTP–labeled T. thermophilus tRNA^Phe transcripts incubated for 30 min at 60°C in the presence of increasing amounts of purified TTC1157. Circles of dotted lines show the migration of the pG, pA, pC, and pU nucleotides used as ultraviolet markers.

FIGURE 5.

In the absence of G6 in tRNA, no m²G is formed by TTC1157. Autoradiography of two-dimensional chromatograms of P1 hydrolysates of [α-³²P] GTP–labeled T. thermophilus tRNA^Phe, tRNA^Asp, and tRNA^Phe(G6C; C67G) transcripts incubated for 30 min at 60°C in the presence of 0.5 μg of purified TTC1157. Circles of dotted lines show the migration of the pA, pC, and pU nucleotides used as ultraviolet markers.

FIGURE 6.

Localization of m²G in tRNA. (Left) Nucleotide sequence of T. thermophilus tRNA^Phe. The arrow points to position 6 of tRNA, and the line indicates the 5-nt fragment isolated after RNase digestion. (Center) Autoradiogram of the 30% PAGE used to separate the RNase A fragments of the modified and unmodified tRNA^Phe transcripts. The obtained 5-, 3-, and 2-nt-long fragments are indicated by arrows. The 5-nt-long fragment indicated by an asterisk corresponds to the lined nucleotides in A. (Right) Autoradiography of two-dimensional chromatograms of T2 hydrolysates of the labeled 5-nt (*) fragment obtained from transcripts of [α-³²P] GTP–labeled T. thermophilus tRNA^Phe incubated for 30 min at 60°C in the presence or absence of purified TTC1157.

FIGURE 7.

Bandshift assay with an [α-³²P] GTP–labeled T. thermophilus tRNA^Phe transcript and PF1002 or the separated MTase and THUMP domains. Increasing amounts of the proteins were used in a binding experiment on [α-³²P] GTP–labeled T. thermophilus tRNA^Phe transcript. The complexes were separated on a 6% PAGE.