Published online 30 April 2004
Nucleic Acids Research, 2004, Vol. 32, No. 8 2453-2463
© 2004 Oxford University Press
Sequence–structure–function studies of tRNA:m5C methyltransferase Trm4p and its relationship to DNA:m5C and RNA:m5U methyltransferases
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Warsaw, Poland and 1 Fort Wayne Center for Medical Education, Indiana University School of Medicine, 2101 Coliseum Boulevard East, Fort Wayne, IN 46805, USA
*To whom correspondence should be addressed. Tel: +1 260 481 6701; Fax: +1 260 481 6408; Email: redman{at}ipfw.edu
Received January 22, 2004; Revised March 16, 2004; Accepted April 2, 2004
Three types of methyltransferases (MTases) generate 5-methylpyrimidine in nucleic acids, forming m5U in RNA, m5C in RNA and m5C in DNA. The DNA:m5C MTases have been extensively studied by crystallographic, biophysical, biochemical and computational methods. On the other hand, the sequence–structure–function relationships of RNA:m5C MTases remain obscure, as do the potential evolutionary relationships between the three types of 5-methylpyrimidine-generating enzymes. Sequence analyses and homology modeling of the yeast tRNA:m5C MTase Trm4p (also called Ncl1p) provided a structural and evolutionary platform for identification of catalytic residues and modeling of the architecture of the RNA:m5C MTase active site. The analysis led to the identification of two invariant residues that are important for Trm4p activity in addition to the conserved Cys residues in motif IV and motif VI that were previously found to be critical. The newly identified residues include a Lys residue in motif I and an Asp in motif IV. A conserved Gln found in motif X was found to be dispensable for MTase activity. Locations of essential residues in the model of Trm4p are in very good agreement with the X-ray structure of an RNA:m5C MTase homolog PH1374. Theoretical and experimental analyses revealed that RNA:m5C MTases share a number of features with either RNA:m5U MTases or DNA:m5C MTases, which suggested a tentative phylogenetic model of relationships between these three classes of 5-methylpyrimidine MTases. We infer that RNA:m5C MTases evolved from RNA:m5U MTases by acquiring an additional Cys residue in motif IV, which was adapted to function as the nucleophilic catalyst only later in DNA:m5C MTases, accompanied by loss of the original Cys from motif VI, transfer of a conserved carboxylate from motif IV to motif VI and sequence permutation.
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