Characterization of Schizosaccharomyces pombe RNA triphospatase Journal Article
| Authors: | Pei, Y.; Schwer, B.; Hausmann, S.; Shuman, S. |
| Article Title: | Characterization of Schizosaccharomyces pombe RNA triphospatase |
| Abstract: | RNA triphosphatase catalyzes the first step in mRNA cap formation which entails the cleavage of the β-γ phosphoanhydride bond of triphosphate-terminated RNA to yield a diphosphate end that is then capped with GMP by RNA guanylyltransferase. Here we characterize a 303 amino acid RNA triphosphatase (Pct1p) encoded by the fission yeast Schizosaccharomyces pombe. Pct1p hydrolyzes the γ phosphate of triphosphate-terminated poly(A) in the presence of magnesium. Pct1p also hydrolyzes ATP to ADP and Pi in the presence of manganese or cobalt (Km = 19 μM ATP; kcat = 67 s-1). Hydrolysis of 1 mM ATP is inhibited with increasing potency by inorganic phosphate I0.5 = 1 mM), pyrophosphate (I0.5 = 0.4 mM) and tripolyphosphate (I0.5 = 30 μM). Velocity sedimentation indicates that Pct1p is a homodimer. Pct1p is biochemically and structurally similar to the catalytic domain of Saccharomyces cerevisiae RNA triphosphatase Cet1p. Mechanistic conservation between Pot1p and Cet1p is underscored by a mutational analysis of the putative metal-binding site of Pct1p. Pct1p is functional in vivo in S.cerevisiae in lieu of Cet1p, provided that it is coexpressed with the S.pombe guanylyltransferase. Pct1p and other yeast RNA triphosphatases are completely unrelated, mechanistically and structurally, to the metazoan RNA triphosphatases, suggesting an abrupt evolutionary divergence of the capping apparatus during the transition from fungal to metazoan species. |
| Keywords: | controlled study; protein expression; unclassified drug; nonhuman; animals; mice; phosphatase; evolution; in vivo study; adenosine diphosphate; rna triphosphatase; acid anhydride hydrolases; amino acid sequence; molecular sequence data; sequence homology, amino acid; kinetics; messenger rna; enzyme analysis; saccharomyces cerevisiae; rna caps; nucleotide sequence; amino acid; catalysis; adenosine triphosphate; cobalt; phosphate; phosphates; hydrolysis; phosphoric monoester hydrolases; genetic conservation; metals; amino acid motifs; schizosaccharomyces; metazoa; schizosaccharomyces pombe; magnesium; fungal proteins; capped rna; rna capping; genetic complementation test; nucleotidyltransferases; manganese; transferase; acid anhydride; metazoon; fungal rna; guanosine phosphate; metal binding; pyrophosphate; diphosphates; rna guanylyltransferase; priority journal; article; artificial gene fusion; sedimentation rate; polyphosphates |
| Journal Title: | Nucleic Acids Research |
| Volume: | 29 |
| Issue: | 2 |
| ISSN: | 0305-1048 |
| Publisher: | Oxford University Press |
| Date Published: | 2001-01-15 |
| Start Page: | 387 |
| End Page: | 396 |
| Language: | English |
| PUBMED: | 11139608 |
| PROVIDER: | scopus |
| PMCID: | PMC29678 |
| DOI: | 10.1093/nar/29.2.387 |
| DOI/URL: | |
| Notes: | Export Date: 21 May 2015 -- Source: Scopus |
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