Wybutosine (yW) is a tricyclic nucleoside with a large side chain found at the 3'-position adjacent to the anticodon of eukaryotic phenylalanine tRNA. yW supports codon recognition by stabilizing codon-anticodon interactions during decoding on the ribosome. To identify genes responsible for yW synthesis from uncharacterized genes of Saccharomyces cerevisiae, we employed a systematic reverse genetic approach combined with mass spectrometry ('ribonucleome analysis'). Four genes YPL207w, YML005w, YGL050w and YOL141w (named TYW1, TYW2, TYW3 and TYW4, respectively) were essential for yW synthesis. Mass spectrometric analysis of each modification intermediate of yW revealed its sequential biosynthetic pathway. TYW1 is an iron-sulfur (Fe-S) cluster protein responsible for the tricyclic formation. Multistep enzymatic formation of yW from yW-187 could be reconstituted in vitro using recombinant TYW2, TYW3 and TYW4 with S-adenosylmethionine, suggesting that yW synthesis might proceed through sequential reactions in a complex formed by multiple components assembled with the precursor tRNA. This hypothesis is also supported by the fact that plant ortholog is a large fusion protein consisting of TYW2 and TYW3 with the C-terminal domain of TYW4.
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| Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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| Site | Modification | Modifier | Source | Reference |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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| Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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| Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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| Evidence ID | Analyze ID | File | Description |
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