The yggH gene of Escherichia coli encodes a tRNA (m7G46) methyltransferase - PubMed (original) (raw)

The yggH gene of Escherichia coli encodes a tRNA (m7G46) methyltransferase

Lara G S De Bie et al. J Bacteriol. 2003 May.

Abstract

We cloned, expressed, and purified the Escherichia coli YggH protein and show that it catalyzes the S-adenosyl-L-methionine-dependent formation of N(7)-methylguanosine at position 46 (m(7)G46) in tRNA. Additionally, we generated an E. coli strain with a disrupted yggH gene and show that the mutant strain lacks tRNA (m(7)G46) methyltransferase activity.

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Figures

FIG. 1.

FIG. 1.

The product of the E. coli yggH ORF catalyzes the formation of m7G in tRNA. (A) SDS-PAGE of the purified YggHH6 protein. Lane 1, molecular mass markers in kilodaltons (Pharmacia Biotech); lane 2, purified protein. The thick and thin arrows indicate the recombinant YggHH6 protein and its minor contaminant, respectively (see the text for details). (B) Autoradiography of a two-dimensional chromatogram of 5′ phosphate nucleotides on a thin-layer cellulose plate. Total tRNA (100 μg) from the methionine-starved P4X-SB25 strain was incubated in a 200-μl reaction mixture containing 50 mM PIPES [piperazine-N,_N_′-bis(2-ethanesulfonic acid)]-Na (pH 7.0), 4 mM MgCl2, 10 μM [_methyl_-14C]AdoMet (53 mCi/mmol), and 0.4 μg of the purified YggHH6 protein. After a 30-min incubation at 37°C, the tRNA was recovered and digested by nuclease P1, and the resulting nucleotides were analyzed as described previously (12).

FIG. 2.

FIG. 2.

In vitro-transcribed E. coli formula imageis a substrate of the YggH MTase. (A) Cloverleaf representation of the nucleotide sequence of E. coli formula image(9). (B) Autoradiography of two-dimensional chromatograms of 5′ and 3′ phosphate nucleotides on thin-layer cellulose plates. [α-32P]GTP-labeled (a and d) or [α-32P]UTP-labeled (b and e) in vitro-transcribed formula imageand [α-32P]GTP-labeled in vitro-transcribed formula image(c and f) (106 cpm) were incubated in the presence (d, e, and f) or absence (a, b, and c) of the YggHH6 protein. The reaction mixture contained 50 mM PIPES-Na (pH 7.0), 4 mM MgCl2, 50 μM AdoMet, and 0.4 μg of the purified YggHH6 protein. After 30 min of incubation at 37°C, the tRNA was recovered and digested by nuclease P1 (a, c, d, and f) or RNase T2 (b and e), and the resulting nucleotides were analyzed as described in the legend to Fig. 1.

FIG. 3.

FIG. 3.

The E. coli RDB1 strain with an inactivated yggH gene lacks tRNA (m7G46) MTase activity. The panels show autoradiography of two-dimensional chromatograms of 5′ phosphate nucleotides on thin-layer cellulose plates. [α-32P]GTP-labeled in vitro-transcribed formula image(106 cpm) was incubated with a crude extract of the DY330 F′ strain (wild type) (a), of the RDB1 strain (b), or of the RDB1/pCR-yggH strain (c). The reaction mixture contained 50 mM PIPES-Na (pH 7.0), 4 mM MgCl2, 50 μM AdoMet, and 100 μg of total protein. After 30 min of incubation at 37°C, the tRNA was recovered and digested by nuclease P1, and the resulting nucleotides were analyzed as described in the legend to Fig. 1.

FIG. 4.

FIG. 4.

Sequence alignment of the representative members of three m7G MTase families specific for tRNA, mRNA, and 16S rRNA: E. coli YggH, S. cerevisiae Abd1p (cap 0 MTase family), and Streptomyces kanamyceticus Kmr (Agr family). Conserved motifs are labeled according to the nomenclature used by Fauman et al. (10). The number of residues omitted for clarity is indicated in parentheses. Conserved AdoMet-binding carboxylate residues are indicated by asterisks, and conserved residues important for the stability of the MTase fold are indicated with pluses.

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