Importance of homodimerization for the in vivo function of yeast RNA triphosphatase

Saccharomyces cerevisiae RNA triphosphatase Cet1 is an essential component of the yeast mRNA capping apparatus. The active site of Cet1 resides within a topologically closed hydrophilic beta -barrel (the triphosphate tunnel) that is supported by a globular hydrophobic core. The homodimeric quaternar y structure of Cet1 is formed by a network of contacts between the partner protomers. By studying the effects of alanine-cluster mutations, we highlig ht the contributions of two separate facets of the crystallographic dimer i nterface to Cet1 function in vivo. One essential facet of the interface ent ails hydrophobic cross dimer interactions of Cys(330) and Val(331) and a cr oss-dimer hydrogen bond of Asp(280) with the backbone amide of Gln(329). Th e second functionally relevant dimer interface involves hydrophobic side-ch ain interactions of Phe(272) and Leu(273). Ala-cluster mutations involving these residues elicited lethal or severe temperature-sensitive phenotypes t hat were suppressed completely by fusion of the mutated triphosphatases to the guanylyltransferase domain of mammalian capping enzyme. The recombinant D279A-D280A and F272A-L273A proteins retained phosphohydrolase activity bu t sedimented as monomers. These results indicate that a disruption of the d imer interface is uniquely deleterious when the yeast RNA triphosphatase mu st function in concert with the endogenous yeast guanylyltransferase. We al so identify key residue pairs in the hydrophobic core of the Cet1 protomer that support the active site tunnel and stabilize the triphosphatase in viv o.