GENETIC, PHYSICAL, AND FUNCTIONAL INTERACTIONS BETWEEN THE TRIPHOSPHATASE AND GUANYLYLTRANSFERASE COMPONENTS OF THE YEAST MESSENGER-RNA CAPPING APPARATUS

We have characterized an essential Saccharomyces cerevisiae gene, CES5 , that when present in high copy, sup presses the temperature-sensitiv e growth defect caused by the ceg1-25 mutation of the yeast mRNA guany lyltransferase (capping enzyme). CES5 is identical to CET1, which enco des the RNA triphosphatase component of the yeast capping apparatus, P urified recombinant Cet1 catalyzes hydrolysis of the gamma phosphate o f triphosphate-terminated RNA at a rate of 1 s(-1). Cet1 is a monomer in solution; it binds with recombinant Ceg1 in vitro to form a Cet1-Ce g1 heterodimer. The interaction of Cet1 with Ceg1 elicits >10 fold sti mulation of the guanylyltransferase activity of Ceg1, This stimulation is the result of increased affinity for the GTP substrate. A truncate d protein, Cet1(201-549), has RNA triphosphatase activity, heterodimer izes with and stimulates Ceg1 in vitro, and suffices when expressed in single copy for cell growth in vivo. The more extensively truncated d erivative Cet1(246-549) also has RNA triphosphatase activity but fails to stimulate Ceg1 in vitro and is lethal when expressed in single cop y in vivo. These data suggest that the Cet1-Ceg1 interaction is essent ial but do not resolve whether the triphosphatase activity is also nec essary, The mammalian capping enzyme Mce1 (a bifunctional triphosphata se-guanylyltransferase) substitutes for Cet1 in vivo. A mutation of th e triphosphatase active-site cysteine of Mce1 is lethal. Hence, an RNA triphosphatase activity is essential for eukaryotic cell growth, This work highlights the potential for regulating mRNA cap formation throu gh protein-protein interactions.