Giardia lamblia RNA cap guanine-N2 methyltransferase (Tgs2).
Tgs1 is the enzyme responsible for converting 7-methylguanosine RNA caps to the 2,2,7-trimethylguanosine cap structures of small nuclear and small nucleolar RNAs. Whereas budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe encode a single Tgs1 protein, the primitive eukaryote Giardia lamblia encodes two paralogs, Tgs1 and Tgs2. Here we show that purified Tgs2 is a monomeric enzyme that catalyzes methyl transfer from AdoMet (K(m) of 6 microm) to m(7)GDP (K(m) of 65 microm; k(cat) of 14 min(-1)) to form m(2,7)GDP. Tgs2 also methylates m(7)GTP (K(m) of 30 microm; k(cat) of 13 min(-1)) and m(7)GpppA (K(m) of 7 microm; k(cat)) of 14 min(-1) but is unreactive with GDP, GTP, GpppA, ATP, CTP, or UTP. We find that the conserved residues Asp-68, Glu-91, and Trp-143 are essential for Tgs2 methyltransferase activity in vitro. The m(2,7)GDP product formed by Tgs2 can be converted to m(2,2,7)GDP by S. pombe Tgs1 in the presence of excess AdoMet. However, Giardia Tgs2 itself is apparently unable to add a second methyl group at guanine-N2. This result implies that 2,2,7-trimethylguanosine caps in Giardia are either synthesized by Tgs1 alone or by the sequential action of Tgs2 and Tgs1. The specificity of Tgs2 raises the prospect that some Giardia mRNAs might contain dimethylguanosine caps.
Hausmann
Stéphane
S
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
Stewart
S
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D002384:Catalysis; D002499:Centrifugation, Density Gradient; D004305:Dose-Response Relationship, Drug; D016829:Giardia lamblia / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry; D005990:Glycerol / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006147:Guanine / Q000737:chemistry; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances / Q000737:chemistry; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection; D014364:Tryptophan / Q000737:chemistry; D012359:tRNA Methyltransferases / Q000737:chemistry / Q000378:metabolism*
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006151:Guanosine; D049452:Green Fluorescent Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; C017778:N(2),N(2),7-trimethylguanosine; D006147:Guanine; D012313:RNA; D006160:Guanosine Triphosphate; D014364:Tryptophan; D008780:Methyltransferases; D012359:tRNA Methyltransferases; C023196:tRNA (guanine(10)-N(2))-dimethyltransferase; D005990:Glycerol