Importance of homodimerization for the in vivo function of yeast RNA triphosphatase Journal Article
| Authors: | Lehman, K.; Ho, C. K.; Shuman, S. |
| Article Title: | Importance of homodimerization for the in vivo function of yeast RNA triphosphatase |
| Abstract: | 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 β-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 crystallographic dimer interface to Cet1 function in vivo. One essential facet of the interface entails hydrophobic cross-dimer interactions of Cys330 and Val 331 and a cross-dimer hydrogen bond of Asp280 with the backbone amide of Gln329. The second functionally relevant dimer interface involves hydrophobic side-chain interactions of Phe272 and Leu273. 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. |
| Keywords: | unclassified drug; promoter region; genetics; nonhuman; molecular genetics; protein conformation; mouse; phenotype; animal; metabolism; mammalia; animals; mice; enzymology; acid anhydride hydrolase; rna triphosphatase; enzyme activity; acid anhydride hydrolases; rna; gene expression regulation; chemistry; amino acid sequence; molecular sequence data; sequence homology, amino acid; hybrid protein; recombinant fusion proteins; saccharomyces cerevisiae; models, molecular; dimerization; chemical structure; yeast; gene dosage; sequence homology; biochemistry; mutagenesis; enzyme active site; gene expression regulation, enzymologic; nucleotidyltransferase; topology; rna capping; molecular stability; nucleotidyltransferases; hydrophobicity; guanylyltransferase; fungal enzyme; saccharomyces; priority journal; article; homodimerzation |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 276 |
| Issue: | 18 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2001-05-04 |
| Start Page: | 14996 |
| End Page: | 15002 |
| Language: | English |
| DOI: | 10.1074/jbc.M100588200 |
| PUBMED: | 11279098 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 21 May 2015 -- Source: Scopus |
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