Resolution by diagonal gel mobility shift assays of multisubunit complexes binding to a functionally important element of the rat growth hormone gene promoter

• Published in: The Journal of biological chemistry Vol. 265; no. 24; pp. 14592 - 14598
• Main Authors: SCHAUFELE, F, CASSILL, J. A, WEST, B. L, REUDELHUBER, T
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25.08.1990
• Subjects: Animals; Base Sequence; Binding Sites; Biological and medical sciences; Cell Nucleus - metabolism; Chromatography, Affinity; Deoxyribonuclease I; Electrophoresis, Gel, Two-Dimensional; Fundamental and applied biological sciences. Psychology; Growth Hormone - genetics; Immunoblotting; Liver - metabolism; Macromolecular Substances; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Rats; Transcription Factors - isolation & purification; Transcription Factors - metabolism; Transcription, Genetic; Transcription. Transcription factor. Splicing. Rna processing; Transfection; Rat; Transcription promoter; STH; Rodentia; Site directed mutagenesis; Footprinting technique; Binding site; Gel retardation technique; Protein hormone; Vertebrata; Mammalia; Gene; Structure activity relation; Transcription factor
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)77343-2

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.