Importance of Homodimerization for thein VivoFunction of Yeast RNA Triphosphatase
Journal of Biological Chemistry
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 ␤-barrel (the triphosphate tunnel) that is supported by a globular hydrophobic core. The homodimeric quaternary structure of Cet1 is formed by a network of contacts between the partner protomers. By studying the effects of alanine-cluster mutations, we highlight the contributions of two separate facets of the
... stallographic dimer interface to Cet1 function in vivo. One essential facet of the interface entails hydrophobic cross-dimer interactions of Cys 330 and Val 331 and a cross-dimer hydrogen bond of Asp 280 with the backbone amide of Gln 329 . The second functionally relevant dimer interface involves hydrophobic side-chain interactions of Phe 272 and Leu 273 . Ala-cluster mutations involving these residues elicited lethal or severe temperature-sensitive phenotypes that 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 but sedimented as monomers. These results indicate that a disruption of the dimer interface is uniquely deleterious when the yeast RNA triphosphatase must function in concert with the endogenous yeast guanylyltransferase. We also identify key residue pairs in the hydrophobic core of the Cet1 protomer that support the active site tunnel and stabilize the triphosphatase in vivo.