Amino acid residues of the Escherichia coli tRNA(m5U54)methyltransferase (TrmA) critical for stability, covalent binding of tRNA and enzymatic activity

Abstract

The Escherichia coli trmA gene encodes the tRNA(m5U54)methyltransferase, which catalyses the formation of m5U54 in tRNA. During the synthesis of m5U54, a covalent 62-kDa TrmA-tRNA intermediate is formed between the amino acid C324 of the enzyme and the 6-carbon of uracil. We have analysed the formation of this TrmA-tRNA intermediate and m5U54 in vivo, using mutants with altered TrmA. We show that the amino acids F188, Q190, G220, D299, R302, C324 and E358, conserved in the C-terminal catalytic domain of several RNA(m5U)methyltransferases of the COG2265 family, are important for the formation of the TrmA-tRNA intermediate and/or the enzymatic activity. These amino acids seem to have the same function as the ones present in the catalytic domain of RumA, whose structure is known, and which catalyses the formation of m5U in position 1939 of E. coli 23 S rRNA. We propose that the unusually high in vivo level of the TrmA-tRNA intermediate in wild-type cells may be due to a suboptimal cellular concentration of SAM, which is required to resolve this intermediate. Our results are consistent with the modular evolution of RNA(m5U)methyltransferases, in which the specificity of the enzymatic reaction is achieved by combining the conserved catalytic domain with different RNA-binding domains.

Figure 1.

Sequence alignment of four RNA(m⁵U)methyltransferases with known biochemical function. The conserved motifs, TRAM domain and amino acid substitutions (asterisk, number and nature of the amino acid) investigated in this work are marked. The translational start of the Trm2p is according to (4).

Figure 2.

The proposed catalytic mechanism of RNA m⁵U methyltransferases.

Figure 3.

Western blot analysis of trmA mutants. Strains MW100 (wt), GB1-41B (trmA4), GB1-5-39 (trmA5), GB1-6-1 (trmA6), GB1-9-6 (trmA9), GB1-10-4 (trmA10), GRB2293 (trmA15), GRB2294 (trmA16) and GRB1648 (trmA17) were used to prepare the protein extracts. The intensities of the 42- and 62-kDa peptides of wild type and of each mutant are expressed as a percentage of the total intensities of these peptides (42 + 62 kDa; total intensity of 100%, which is not shown). The row labelled ‘TrmA,%wt’ shows the level of total TrmA-associated peptides in the various mutants relative to the level found in wild type. The ‘x’ band was used as an internal control for the amount of the loaded protein extracts. The row labelled ‘m⁵U,%wt (HPLC)’ shows the amount of m⁵U in total tRNA in each mutant expressed as a percentage of the level of m⁵U in tRNA from the wild-type strain. The asterisk indicates that the amount of m⁵U from the HPLC chromatogram is an overestimate due to impurities in the peak.

Figure 4.

Western blot analysis of trmA mutants in motif IV. Strains MW100 (wt), GRB2276 (trmA14) and GRB2230 (trmA18) were used to prepare the protein extracts. The values were calculated as in Figure 3.

Figure 5.

Interactions between amino acid chains, the target uridine and SAH in the active site of RumA. Several selected amino acids in the active site are divided into (A) and (B) for better representation. Corresponding residues in the TrmA are shown within the parenthesis. The image was created with the SwissPDB Viewer program (40) http://www.expasy.org/spdbv/) using the coordinates of RumA-RNA-SAH complex (2BH2) (11) from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). Putative hydrogen bonds are represented by dashed lines.