same

different

Experimental object: Gal4 activator of eukaryotic cells, which can activate the transcription of the galactose gene of Saccharomyces cerevisiae

① Domain exchange experiment: The protein obtained by expressing the fragment of the Gal4 gene (N-terminal 1/3) can bind to DNA normally, but cannot activate the transcription process. Such a protein contains a DNA-binding domain but lacks an activation domain, so the DNA-binding and transcriptional activation domains of Gal4 can function separately.

② Yeast two-hybrid experiment: Construct a hybrid gene that fuses the C-terminal 2/3 sequence encoding the Gal4 protein with the DNA binding domain of the bacterial inhibitor protein LexA. The resulting protein is expressed in yeast with the same reporter plasmid. There is a LexA binding site upstream of the plasmid Gal4 promoter, and the fusion protein can activate the transcription of this reporter. Experiments show that activation is not only mediated by binding to DNA. On the contrary, the role of the DNA-binding domain is only to bind the activation domain. associated with the promoter.

① Homotype domain proteins: A type of protein with a helix-turn-helix motif DNA-binding domain that binds to DNA in the same way as bacterial regulatory proteins

②Zinc-containing DNA binding domain

③Leucine zipper domain: This protein contains both dimerization surface and DNA binding surface. Dimerization: interaction with the hydrophobic surface (leucine) of the alpha helix; DNA binding: interaction with the symmetric DNA recognition site, ultimately clamping on the DNA like a clamp

④Helix-loop-helix domain (HLH): The extended α-helical region in each monomer is embedded in the major groove of DNA. It is similar to the structure of leucine zipper and can also form dimers and is called basic zipper/base. sexual HLH protein

⑤ High-speed motility protein (HMG): The highly conserved peptide chain AY hooks can interact with the minor groove of the DNA helix and significantly change the conformation of the DNA double helix.

with uncertainty

Classification: ①Acidic activation zone (most common, Gal4) ②Glutamine-rich zone (SP1) ③Proline-rich zone (CTF1)

No allosteric activation in eukaryotes

Recruitment

action at a distance

synergy

Combination control

Competition: Competition with the activator for the binding site. By binding to overlapping sites that bind activators, repressors inhibit the binding of activators to genes, thereby blocking gene activation.

Inhibition: Inhibits activator function. The inhibitor binds to a site next to the activator and interacts with the activator, sterically blocking its activation region.

Direct inhibition: competes with activator for binding to polymerase. The inhibitory factor binds to the site upstream of the gene and inhibits the initiation of transcription by acting in a special way with the transcription apparatus.

Indirect inhibition: recruitment of nucleosome modifiers. Represses transcription by recruiting histone-modifying enzymes to alter nucleosomes. Such as methylation modification and deacetylase action.

Examples of signaling pathways

Signaling pathway: The initial signal/ligand binds to the extracellular domain of the specific receptor on the cell surface → the signal is transmitted to the intracellular domain of the receptor (through allostery or dimerization of the receptor) → the signal is then transmitted in a relay to the relevant transcriptional regulator → the transcriptional regulator controls the expression of the target gene

Mechanisms by which signals control the activity of transcriptional regulators in eukaryotic cells