Functional Analysis of Composite Elements
List of experiments that are used to confirm cooperative interactions between transcription factors within composite elements and conclusions that can be drawn from the particular experiment.
| Identifier | Type of Experiment | Conclusion Drawn from Experiment |
|---|---|---|
| 1.0 | Functional synergism between sites | |
| 1.1 | Site-directed mutagenesis and study of promoter activity | Functional synergism between sites |
| 1.2 | Insertion or deletion of spacer nucleotides between sites and study of promoter activity | Spacing between the sites is important |
| 1.3 | The composite element is shown to transfer specific properties to a heterologous promoter | Functional synergism between sites |
| 2.1 | Site-directed mutagenesis and study of promoter activity | Functional antagonism between sites |
| 3.1 | Transient co-transfection | Functional synergism between factors |
| 3.2 | Stable co-transfection | |
| 3.3 | Co-transfection of plasmids expressing mutated derivatives of the factor(s) | |
| 4.1 | Co-transfection | Functional antagonism between sites |
| 4.2 | Co-transfection of plasmids expressing mutated derivatives of the factor(s) | |
| 5.1 | Co-transfection experiment combined with site-directed mutagenesis | Both intact sites as well as both factors are essential for synergism |
| 6.0 | Cooperative binding of factors to DNA | |
| 6.1 | Gel mobility shift combined with site-directed mutagenesis | |
| 6.2 | Methylation interference assay | |
| 6.3 | DNAse I footprinting analysis | |
| 6.4 | Gel mobility shift in the presence of oligonucleotide competitors | Cooperative binding of factors to DNA, ternary complex formation |
| 6.41 | Gel mobility shift in the presence of oligonucleotide competitors | Cooperative binding of factors to DNA, higher-order complex formation |
| 6.5 | Gel mobility shift in the presence of a single factor or both factors together | Cooperative binding of factors to DNA, ternary complex formation |
| 6.51 | Gel mobility shift in the presence of a single factor or both factors together | Cooperative binding of factors to DNA, higher-order complex formation |
| 6.6 | Cu2+-phenanthroline footprinting assay | Cooperative binding of factors to DNA, ternary complex formation |
| 6.7 | in vivo chromatin immunoprecipitation assay (CHIP) | Cooperative binding of factors to DNA |
| 6.8 | Quantitative analysis of DNA binding | |
| 6.9 | DNA affinity binding assay combined with site-directed mutations | |
| 7.0 | Competition between TFs for overlapping binding sites | |
| 7.1 | Electrophoretic mobility shift assay (EMSA) | Competition for overlapping binding sites |
| 8.0 | Mutually exclusive binding of factors to DNA | |
| 8.1 | Gel mobility shift | |
| 8.2 | Methylation interference assay | |
| 9.0 | Ternary complex formation between two factors and DNA | |
| 9.1 | Gel mobility shift using increasing amounts of one of the factors | |
| 9.2 | Study of half-dissociation time of the complexes | |
| 9.21 | Study of half-dissociation time of the complexes | High-order complex formation between factors and DNA |
| 9.3 | Inspection of protein-DNA complex with antibodies | Ternary complex formation |
| 9.31 | Inspection of protein-DNA complex with antibodies | Higher-order complex formation |
| 9.4 | Gel mobility shift combined with mutational analysis of the factor(s) | Ternary complex formation |
| 9.41 | Gel mobility shift combined with mutational analysis of the factor(s) | Higher-order complex formation |
| 10.0 | Direct factor-factor interactions in the absence of DNA | |
| 10.1 | in vitro cross-linking between factors | |
| 10.2 | Mutational analysis of factor(s) | |
| 10.3 | in vivo cross-linking between factors | |
| 11.1 | Over-expression of a co-activator restores transcription level | Competition between TFs for a transcriptional co-activator |
| 11.2 | in vivo cross-linking between factors and co-activator | Simultaneous interactions of factors with co-activator |
| 12.1 | Fluorescence architectural analysis | Cooperative DNA bending |
| 13.1 | Crystallization | Detailed structure of a ternary complex |