In this article, we have described methods used to purify Rnt1p and study its biochemical properties. Rnt1p can be easily purified from bacteria as N-terminal His6-tagged protein and its activity may be monitored in vitro. Rnt1p cleaves the RNA by binding to a cleavage site followed by hydrolysis and product release. The kinetic parameters of Rnt1p are similar to those of other nucleases, including bacterial RNase III. The ability of Rnt1p to bind substrate without cleaving it in the absence of divalent metal ions provides a convenient means to study RNA recognition and binding independent of catalysis. The gel mobility shift and in-the-gel cleavage assays described here reveal the formation of two Rnt1p-RNA complexes with different cleavage activities, suggesting that the protein may bind the substrate in two different forms or through a two-step binding reaction.
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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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