真核生物基因表达调控.pptVIP

真核生物基因表达调控 (Activation domain is interchangeable) Interaction Assays Design of Two-hybrid / Three-hybrid /etc… separable functional domains Two-hybrid assay (protein-protein) Tri-hybrid assay (protein-RNA) 1. RNA polymerase II2. promoter and enhancers3. transcription factors Eukaryotic gene expression is usually controlled at the level of initiation of transcription. 真核基因转录起始的调控 Holoenzyme --- a supramolecular complex comprising Pol II, most GTFs, and Mediator/Srb complex In yeast, a 2MDa holoenzyme + TBP suffices for transcription Ordered Assembly and Pol II Holoenzyme TFIID TFIID one-step multiple-step TFIIB binds to DNA and contacts RNA polymerase near the RNA exit site and at the active center, and orients it on DNA. +25bp Q: prok -10bp vs euk -25bp? Sequential Assembly TBP: TATA binding protein TAFs: TBP associated factors Binding of TFIID (TBP + 11 TAFs, 800KD) to the TATA box is the first step in initiation. CTD：RNA Pol II C-terminal domain CTD is an unusual extension appended to the C terminus of the largest subunit of RNA polymerase II. It comprises from 25 to 52 tandem copies of the consensus repeat heptad Y1S2P3T4S5P6S7. S2 and S5 are major phosphorylation sites. CTD phosphorylation cause the conversion of proline isomerization states. Phosphorylation patterns on the CTD repeats determine different sets of associated factors, so that provide a dynamic platform to recruit different regulators of the transcription apparatus. In eukaryotic cells, the transcription of genes is accurately orchestrated both spatially and temporally by the C-terminal domain of RNA polymerase II (CTD). PIC: PhosphoS5 is required for assembly of the PIC and facilitates mRNA capping via recruitment of capping enzymes. Elongation: S5 gradually becomes dephosphorylated, whereas S2 is phosphorylated. Terminating: PhosphoS2 ensures efficient 3′-RNA processing by triggering recruitment of 3′-RNA processing machinery. Ending: CTDs are free of phosphate groups; non-ph