The Ski2-like RNA helicase, Mtr4, plays a central role in nuclear RNA surveillance pathways by delivering targeted substrates to the RNA exosome for processing or degradation. RNA target selection is accomplished by a variety of Mtr4-mediated protein complexes. In S. cerevisiae, the Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complex prepares substrates for exosomal decay through the combined action of polyadenylation and helicase activities. Biophysical and structural studies of Mtr4 and TRAMP require highly purified protein components. Here, we describe robust protocols for obtaining large quantities of pure, active Mtr4 and Trf4-Air2 from S. cerevisiae. The proteins are recombinantly expressed in E. coli and purified using affinity, ion exchange, hydrophobic exchange and size exclusion chromatography. Care is taken to remove nuclease contamination during the prep. Assembly of TRAMP is achieved by combining individually purified Mtr4 and Trf4-Air2. We further describe a strand displacement assay to characterize Mtr4 helicase unwinding activity.
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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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Increase the total number of rows showing on this page by using the pull-down located below the table, or use the page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i" buttons located within a cell for an annotation to view further details about experiment type and any other genes involved in the interaction.
| 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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