An essential surface motif (WAQKW) of yeast RNA triphosphatase mediates formation of the mRNA capping enzyme complex with RNA guanylyltransferase

Saccharomyces cerevisiae RNA triphosphatase (Cet1p) and RNA guanylyltransfe rase (Ceg1p) interact in vivo and in vitro to form a bifunctional mRNA capp ing enzyme complex. Cet1p binding to Ceg1p stimulates the guanylyltransfera se activity of Ceg1p. Here we localize the guanylyltransferase-binding and guanytyltransferase-stimulation functions of Cet1p to a 21-amino acid segme nt from residues 239 to 259. The guanylyltransferase-binding domain is loca ted on the protein surface, as gauged by protease sensitivity, and is conse rved in the Candida albicans RNA triphosphatase CaCet1p. Alanine-cluster mu tations of a WAQKW motif within this segment abolish guanylyltransferase-bi nding in vitro and Cet1p function in vivo, but do not affect the triphospha tase activity of Cet1p. Proteolytic footprinting experiments provide physic al evidence that Cet1p interacts with the C-terminal domain of Ceg1p. Tryps in-sensitive sites of Ceg1p that are shielded from proteolysis when Ceg1p i s bound to Cet1p are located between nucleotidyl transferase motifs V and V I.