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 guanylyltransferase (Ceg1p) interact in vivo and in vitro to form a bifunctional mRNA capping enzyme complex. Cet1p binding to Ceg1p stimulates the guanylyltransferase activity of Ceg1p. Here we localize the guanylyltransferase-binding and guanylyltransferase-stimulation functions of Cet1p to a 21-amino acid segment from residues 239 to 259. The guanylyltransferase-binding domain is located on the protein surface, as gauged by protease sensitivity, and is conserved in the Candida albicans RNA triphosphatase CaCet1p. Alanine-cluster mutations of a WAQKW motif within this segment abolish guanylyltransferase-binding in vitro and Cet1p function in vivo, but do not affect the triphosphatase activity of Cet1p. Proteolytic footprinting experiments provide physical evidence that Cet1p interacts with the C-terminal domain of Ceg1p. Trypsin-sensitive sites of Ceg1p that are shielded from proteolysis when Ceg1p is bound to Cet1p are located between nucleotidyl transferase motifs V and VI.
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Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Lehman
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Shuman
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D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000409:Alanine; D000595:Amino Acid Sequence; D002176:Candida albicans / Q000201:enzymology / Q000235:genetics; D002846:Chromatography, Affinity; D017124:Conserved Sequence; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000098556:mRNA Guanylyltransferases
D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase; D000409:Alanine