Specificity and mechanism of RNA cap guanine-N2 methyltransferase (Tgs1) Journal Article
| Authors: | Hausmann, S.; Shuman, S. |
| Article Title: | Specificity and mechanism of RNA cap guanine-N2 methyltransferase (Tgs1) |
| Abstract: | The 2,2,7-trimethylguanosine (TMG) cap structure is characteristic of certain eukaryotic small nuclear and small nucleolar RNAs. Prior studies have suggested that cap trimethylation might be contingent on cis-acting elements in the RNA substrate, protein components of a ribonucleoprotein complex, or intracellular localization of the RNA substrate. However, the enzymatic requirements for TMG cap formation remain obscure because TMG synthesis has not been reconstituted in vitro from defined components. Tgs1 is a conserved eukaryal protein that was initially identified as being required for RNA cap trimethylation in vivo in budding yeast. Here we show that purified recombinant fission yeast Tgs1 catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to m7GTP and m7GDP. Tgs1 also methylates the cap analog m7GpppA but is unreactive with GTP, GDP, GpppA, m2,2,7GTP, m2,2,7GDP, ATP, CTP, UTP, and ITP. The products of methyl transfer to m7GTP and m7GDP formed under conditions of excess methyl acceptor are 2,7-dimethyI GTP and 2,7-dimethyl GDP, respectively. Under conditions of limiting methyl acceptor, the initial m2,7GDP product is converted to m2,2,7GDP in the presence of excess AdoMet. We conclude that Tgs1 is guanine-specific, that N7 methylation must precede N2 methylation, that Tgs1 acts via a distributive mechanism, and that the chemical steps of TMG synthesis do not require input from RNA or protein cofactors. |
| Keywords: | controlled study; unclassified drug; methylation; nonhuman; proteins; protein; protein binding; in vivo study; in vitro study; dose-response relationship, drug; recombinant enzyme; dna methylation; time factors; rna; methyltransferase; methyltransferases; guanine; guanosine derivative; guanosine; recombinant proteins; cellular distribution; yeast; catalysis; adenosine triphosphate; enzyme kinetics; small nucleolar rna; rna, small nucleolar; enzyme specificity; enzyme substrate complex; guanosine triphosphate; synthesis (chemical); enzyme mechanism; cis acting element; schizosaccharomyces; dna, complementary; schizosaccharomyces pombe; trimethylation; s-adenosylmethionine; guanosine diphosphate; capped rna; rna capping; rna methylation; s adenosylmethionine; methyl group; 2,2,7 trimethylguanosine; small nuclear rna; cytidine triphosphate; ribonucleoprotein; nucleotide metabolism; guanine n2 methyltransferase; uridine triphosphate; structure (composition); methyl transfer; protein cofactors; inosine triphosphate; chromatography, thin layer; dithiothreitol |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 280 |
| Issue: | 6 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2005-02-11 |
| Start Page: | 4021 |
| End Page: | 4024 |
| Language: | English |
| DOI: | 10.1074/jbc.C400554200 |
| PUBMED: | 15590684 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 21" - "Export Date: 24 October 2012" - "CODEN: JBCHA" - "Source: Scopus" |
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