Amino acid residues of the Escherichia coli tRNA(m5U54)methyltransferase (TrmA) critical for stability, covalent binding of tRNA and enzymatic activity.
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.
Urbonavicius
Jaunius
J
Department of Molecular Biology, Umeå University, S-90187 Umeå, Sweden.
Jäger
Gunilla
G
Björk
Glenn R
GR
D000595:Amino Acid Sequence; D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013941:Thymine / Q000378:metabolism; D012359:tRNA Methyltransferases / Q000737:chemistry
D029968:Escherichia coli Proteins; C520618:TrmA protein, E coli; D012343:RNA, Transfer; D012359:tRNA Methyltransferases; C022769:tRNA(uracil-5)-methyltransferase; D013941:Thymine