The RNA:pseudouridine (Psi) synthase Pus7p of Saccharomyces cerevisiae is a multisite-specific enzyme that is able to modify U(13) in several yeast tRNAs, U(35) in the pre-tRNA(Tyr) (GPsiA), U(35) in U2 small nuclear RNA, and U(50) in 5 S rRNA. Pus7p belongs to the universally conserved TruD-like family of RNA:Psi-synthases found in bacteria, archaea, and eukarya. Although several RNA substrates for yeast Pus7p have been identified, specificity of their recognition and modification has not been studied. However, conservation of a 7-nt-long sequence, including the modified U residue, in all natural Pus7p substrates suggested the importance of these nucleotides for Pus7p recognition and/or catalysis. Using site-directed mutagenesis, we designed a set of RNA variants derived from the yeast tRNA(Asp)(GUC), pre-tRNA(Tyr)(GPsiA), and U2 small nuclear RNA and tested their ability to be modified by Pus7p in vitro. We demonstrated that the highly conserved U(-2) and A(+1) residues (nucleotide numbers refer to target U(0)) are crucial identity elements for efficient modification by Pus7p. Nucleotide substitutions at other surrounding positions (-4, -3, +2, +3) have only a moderate effect. Surprisingly, the identity of the nucleotide immediately 5' to the target U(0) residue (position -1) is not important for efficient modification. Alteration of tRNA three-dimensional structure had no detectable effect on Pus7p activity at position 13. However, our results suggest that the presence of at least one stem-loop structure including or close to the target U nucleotide is required for Pus7p-catalyzed modification.
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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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