Wild type GAL4 binds cooperatively to the GAL1-10 UASG in vitro

摘要: Transcription of the genes required for utilization galactose in Saccharomyces cerevisiae is controlled primarily by transcriptional activator protein GAL4. The upstream activating sequences (UASG) most GAL have multiple sites to which GAL4 can bind. In this report we compare binding properties wild type and derivatives bearing N-terminal DNA-binding domain vitro. To produce GAL4, constructed a recombinant baculovirus expression insect cells. Recombinant was found bind efficiently an oligonucleotide containing near-consensus 17-mer site electrophoretic mobility shift assays. Footprinting experiments revealed that binds cooperatively four GAL1-10 UASG; however, contrast fragment only DNA-binding/dimerization domains each these with slightly different affinity. With increasing concentrations GAL4(1-147), become filled following order: II, IV, I, III. fully occupied at approximately same concentration protein. footprints on USAG, enhancements protections DNase I-sensitive cleavages are detectable between III indicative formation loop distantly spaced sites. Binding strong assists adjacent mutant both footprinting GAL4(1-147) fused portions GAL4's region II were incapable cooperative DNA our We conclude from observations has function distinct dimerization or activation functions, likely plays important role precise regulation gene transcription.