activating Domain swap region Gal4 DNA-binding experiment domain Moving domains Gal4 site among proteins proving that domains lacz OFF can be dissected Gal4 site into separate parts f the proteins DNA-binding domain Many similar acz OFF experiments LexA site shows that dNa activating region binding domains LexA DNA- binding and activating domain ON regions are LexA sit 2004 Pearson Education, Inc, publishing as Benjamin separable
17 Domain swap experiment Moving domains among proteins, proving that domains can be dissected into separate parts of the proteins. Many similar experiments shows that DNA binding domains and activating regions are separable
Box1 The two hybrid assay(酵母双杂交)is used to identify proteins interacting with each other.(实验介绍系列2) activating region gene NO transcription DNA-binding site Fuse protein A and protein B genes to the dNa binding domain and activating region of DNA-binding domain Gal4, respectively gene NO transcription B Produce fusion proteins gen transcription DNA-binding site L8
18 Box 1 The two hybrid Assay (酵母双杂交) is used to identify proteins interacting with each other. （实验介绍系列2） Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4, respectively. Produce fusion proteins
2. Eukaryotic regulators use a range of dna binding domains, but DNA recognition involves the same principles as found in bacteria a Homeodomain proteins Zinc containing DNA-binding domain zinc finger and zinc cluster a Leucine zipper motif a Helix-Loop-Helix proteins basic zipper and HLh proteins 19
19 2. Eukaryotic regulators use a range of DNA binding domains, but DNA recognition involves the same principles as found in bacteria. ◼ Homeodomain proteins ◼ Zinc containing DNA-binding domain: zinc finger and zinc cluster ◼ Leucine zipper motif ◼ Helix-Loop-Helix proteins : basic zipper and HLH proteins
Bacterial regulatory proteins Most use the helix-turn-helix motif to bind DNA target Most bind as dimers to DNA sequence each monomer inserts an a helix into the maior groove Eukaryotic regulatory proteins 1. Recognize the DNA using the similar rinciples, with some variations in detail 2. In addition to form homodimers(同源二聚 体, some form heterodimers(异源二聚体) to recognize DNA, extending the range of DNA-binding specificity
20 Bacterial regulatory proteins • Most use the helix-turn-helix motif to bind DNA target • Most bind as dimers to DNA sequence: each monomer inserts an a helix into the major groove. Eukaryotic regulatory proteins 1. Recognize the DNA using the similar principles, with some variations in detail. 2. In addition to form homodimers (同源二聚 体), some form heterodimers (异源二聚体) to recognize DNA, extending the range of DNA-binding specificity
Homeodomain proteins: The homeodomain(F 源结构域) is a class of helix-urn-heix DNA-binding domain and recognizes DNA in essentially the same way as those bacterial proteins What is the same? Ser 2 rg Asn srg Figure 17-5 21
21 Homeodomain proteins: The homeodomain (同 源结构域) is a class of helix-turn-helix DNA-binding domain and recognizes DNA in essentially the same way as those bacterial proteins. Figure 17-5 What is the same?