Abstract
Cet1, the RNA triphosphatase component of the yeast mRNA capping apparatus, catalyzes metal-dependent gamma phosphate hydrolysis within the hydrophilic interior of a topologically closed 8-strand beta barrel (the "triphosphate tunnel"). We used structure-guided alanine scanning to identify 6 side chains within the triphosphate tunnel that are essential for phosphohydrolase activity in vitro and in vivo: Arg393, Glu433, Arg458, Arg469, Asp471 and Thr473. Alanine substitutions at two positions, Asp377 and Lys409, resulted in partial catalytic defects and a thermosensitive growth phenotype. Structure-function relationships were clarified by introducing conservative substitutions. Five residues were found to be nonessential: Lys309, Ser395, Asp397, Lys427 Asn431, and Lys474. The present findings, together with earlier mutational analyses, reveal an unusually complex active site in which 15 individual side chains in the tunnel cavity are important for catalysis, and each of the 8 strands of the beta barrel contributes at least one functional constituent. The active site residues fall into three classes: (i) those that participate directly in catalysis via coordination of the gamma phosphate or the metal; (ii) those that make critical water-mediated contacts with the gamma phosphate or the metal; and (iii) those that function indirectly via interactions with other essential side chains or by stabilization of the tunnel structure.
Highlights
Evisiae, Candida albicans, and Schizosaccharomyces pombe are strictly dependent on a divalent cation (5–10)
The present findings, together with earlier mutational analyses, reveal an unusually complex active site in which 15 individual side chains in the tunnel cavity are important for catalysis, and each of the 8 strands of the  barrel contributes at least one functional constituent
The active site residues fall into three classes: (i) those that participate directly in catalysis via coordination of the ␥ phosphate or the metal; (ii) those that make critical water-mediated contacts with the ␥ phosphate or the metal; and (iii) those that function indirectly via interactions with other essential side chains or by stabilization of the tunnel structure
Summary
Expression and Purification of Mutated Versions of Yeast RNA Triphosphatase—Missense mutations were introduced into the CET1(201–549) gene by polymerase chain reaction by using the twostage overlap extension method (18). The peak fractions were pooled, adjusted to 5 mM EDTA, and dialyzed against buffer C (50 mM Tris-HCl, pH 8.0, 2 mM DTT, 5 mM EDTA, 10% glycerol, 0.05% Triton X-100) containing 50 mM NaCl. Protein concentrations were determined by the Bio-Rad dye binding method with bovine serum albumin as the standard. RNA Triphosphatase Assay—Reaction mixtures (10 l) containing 50 mM Tris-HCl, pH 7.5, 5 mM DTT, 1 mM MgCl2, 20 pmol of 5Ј [␥-32P]poly(A), and enzyme were incubated for 15 min at 30 °C. Mutational Effects on RNA Triphosphatase Function in Vivo—NdeI/ BamHI fragments encoding mutated versions of Cet1(201–549) were excised from the respective pET16b-CET1 plasmids and inserted into the yeast CEN TRP1 plasmid pCET1–5Ј3Ј (5) so that expression of the inserted gene is under the control of the natural CET1 promoter. Growth was assessed as follows: ϩϩ indicates wild-type colony size at all temperatures, and ts indicates growth at 16, 22, and 30 °C but no growth at 37 °C (Table I)
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