Resolution by diagonal gel mobility shift assays of multisubunit complexes binding to a functionally important element of the rat growth hormone gene promoter
| Autor: | F, Schaufele, J A, Cassill, B L, West, T, Reudelhuber |
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| Rok vydání: | 1990 |
| Předmět: |
Cell Nucleus
Binding Sites Base Sequence Transcription Genetic Macromolecular Substances Immunoblotting Molecular Sequence Data Transfection Chromatography Affinity Rats Liver Growth Hormone Mutation Animals Deoxyribonuclease I Electrophoresis Gel Two-Dimensional Promoter Regions Genetic Transcription Factors |
| Zdroj: | The Journal of biological chemistry. 265(24) |
| ISSN: | 0021-9258 |
| Popis: | DNase I footprinting identifies a tissue-general factor, GHF3, binding to the rat growth hormone promoter between nucleotides -239 and -219. Mutation of the GHF3-binding site reduces promoter activity to 30% of that of the wild-type promoter after transfection into GC cells. Southwestern blotting and protein/DNA cross-linking experiments demonstrate that the GHF3-binding factor migrates as a 43-kDa protein. However, multiple GHF3 factor/DNA complexes with different electrophoretic mobilities are detected by gel retardation analysis. A novel technique, the diagonal gel mobility shift assay, is used to demonstrate that five of the different complexes represent multisubunit structures containing a common DNA-binding subunit. In this method, the multisubunit complexes resolved by one-dimensional gel mobility shift assays are observed to partially dissociate during electrophoresis in a second dimension with the DNA-binding subunit detected as a common signal directly below those signals representing the undissociated complexes which lie on a diagonal line. Two of the five complexes also contain an additional subunit in common whereas two other complexes appear to contain completely different subunits interacting with the common DNA-binding subunit. All five complexes copurify during GHF3-binding site-specific DNA affinity chromatography, and this fraction stimulates in vitro transcription in a GHF3-binding site-dependent fashion. Thus, a functionally important region of the rat growth hormone gene promoter interacts with a DNA-binding transcription factor which in turn acts as a docking site for other proteins. |
| Databáze: | OpenAIRE |
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