Polyamines are critically important for protein synthesis. Through their positive ionic charge, polyamines readily bind to ribosomes, as well as to mRNAs and tRNAs. Moreover, the polyamine spermidine serves as a substrate for the synthesis of hypusine, an essential post-translational modification on the translation factor eIF5A. Though originally thought to function in translation initiation, eIF5A is now known to generally promote translation elongation and termination. Moreover, translation of certain motifs like polyproline show a greater dependency on eIF5A. In this chapter, we describe the biochemical assays we use to study eIF5A and its regulation. Owing to the complex nature of protein synthesis, these assays require the purification of over 10 translation factors plus ribosomes, tRNAs, and aminoacyl-tRNA synthetases. We describe the methods used to purify these components, to synthesize the mRNA templates for translation, and to resolve the translation products by electrophoretic thin-layer chromatography. With the recent identification of eIF5A as a key target for regulating the synthesis of polyamine synthesis and transport, and the recent identification of mutations in eIF5A causing a neurodevelopmental disorder, the assays described in this chapter will be useful in further elucidating the function and regulation of this enigmatic protein.
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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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