Mutational analyses of yeast RNA triphosphatases highlight a common mechanism of metal-dependent NTP hydrolysis and a means of targeting enzymes to pre-mRNAs in vivo by fusion to the guanylyltransferase component of the capping apparatus

Saccharomyces cerevisiae Cet1p is the prototype of a family of metal-depend ent RNA 5'-triphosphatases/ NTPases encoded by fungi and DNA viruses; the f amily is defined by conserved sequence motifs A, B, and C, We tested the ef fects of 12 alanine substitutions and 16 conservative modifications at 18 p ositions of the motifs, Eight residues were identified as important for tri phosphatase activity. These were Glu-305, Glu-307, and Phe310 in motif A (I ELEMKF); Arg-454 and Lys-456 in motif B (RTK); Glu-492, Glu-494, and Glu-49 6 in motif C (EVELE), Four acidic residues, Glu-305, Glu-307, Glu-494, and Glu-496, may comprise the metal-binding site(s), insofar as their replaceme nt by glutamine inactivated Cet1p, E492Q retained triphosphatase activity. Basic residues Arg-454 and Lys-456 in motif B are implicated in binding to the 5'-triphosphate, Changing Arg-454 to alanine or glutamine resulted in a 30-fold increase in the K-m for ATP, whereas substitution with lysine incr eased K-m 6-fold. Changing Lys-456 to alanine or glutamine increased K-m an order of magnitude; ATP binding was restored when arginine was introduced. Alanine in lieu of Phe-310 inactivated Cet1p, whereas Tyr or Leu restored function, Alanine mutations at aliphatic residues Leu-306, Val-493, and Leu -495 resulted in thermal instability in vivo and in vitro. A second S. cere visiae RNA triphosphatase/NTPase (named Cth1p) containing motifs A, B, and C was identified and characterized. Cth1p activity was abolished by E87A an d E89A mutations in motif A. Cth1p is nonessential for yeast growth and, by itself, cannot fulfill the essential role played by Cet1p in vivo, Yet, fu sion of Cth1p in cis to the guanylyltransferase domain of mammalian capping enzyme allowed Cth1p to complement growth of cet1 Delta yeast cells. This finding illustrates that mammalian guanylyltransferase can be used as a veh icle to deliver enzymes to nascent pre-mRNAs in vivo, most likely through i ts binding to the phosphorylated CTD of RNA polymerase II.