Yeast and viral RNA 5 ' triphosphatases comprise a new nucleoside triphosphatase family

Saccharomyces cerevisiae Cet1p catalyzes the first step of mRNA capping, th e hydrolysis of the gamma phosphate of triphosphate-terminated RNA to form a 5' diphosphate end. The RNA triphosphatase activity of Cet1p is magnesium -dependent and has a turnover number of 1 s(-1), Here we show that purified recombinant Cet1p possesses a robust ATPase activity (K-m = 2.8 mu M; V-ma x = 25 s(-1)) in the presence of manganese. Cobalt is: also an effective co factor, but magnesium, calcium, copper, and zinc are not. Cet1p displays br oad specificity in converting ribonucleoside triphosphates and deoxynucleos ide triphosphates to their respective diphosphates. The manganese- and coba lt-dependent nucleoside triphosphatase of Cet1p resembles the nucleoside tr iphosphatase activities of the baculovirus LEF-4 and vaccinia virus D1 capp ing enzymes. Cet1p, LEF-4, and D1 share three collinear sequence motifs, Mu tational analysis establishes that conserved glutamate and arginine: side c hains within these motifs are essential for the RNA triphosphatase and ATPa se activities of Cet1p in vitro and for Cet1p function in vivo. These findi ngs are in accord with the effects of single alanine mutations at analogous positions of vaccinia capping enzyme. We suggest that the metal-dependent RNA triphosphatases encoded by yeast and DNA viruses comprise a novel famil y of phosphohydrolase enzymes with a common active site.