Section N Regulation of Transcription in Eukaryotes

1. Section N Regulation of Transcription in Eukaryotes N1 Eukaryotic Transcription FactorsN2 Examples of Transcriptional Regulation Yang Xu, College of Life Sciences

2. N1 Eukaryotic Transcription Factors • Structure of a typical eukaryotic gene • Transcription factor domain structure • DNA-binding domains • Dimerization domains • Transcription activation • Repressor domains • Targets for transcriptional regulation Yang Xu, College of Life Sciences

3. Structure of a typical eukaryotic gene Yang Xu, College of Life Sciences

4. Transcription factor domain structure Specific trans-factor characteristic DNA-binding domain activation domain 3. Dimerization domain (in some dimer factor) 1. DNA-binding domain 2. Activation domain

5. DNA-binding domains Consist of: • Helix-turn-Helix: (a 60aa homeodomain) • Zinc fingerdomain: (C2H2 and C4zinc finger) • Basic domain: (bZIP or bHLH) Yang Xu, College of Life Sciences

6. Helix Turn Recognition helix DNA Helix-turn-helix domain Structure: a 60aa homeodomain encoded by the homeobox. Found in: • Antennapedia TF of Dropphila • Phage DNA-binding proteins such as the l cro repressor; • Lac and trp repressors; • cAMP receptor protein, CRP. DNA-binding domain: • Recognition helix, lies partly in the major groove and interacts with the DNA. Yang Xu, College of Life Sciences

7. Helix-turn-helix domain and binding with DNA Recognition helix 2 2 3 3 1 1

8. Peptide chain DNA binding sites R R H R C H C H Zn Zn C H C H C N C C C C Zn Zn C C C C N C Zinc fingerdomain-I C2H2 zinc finger: TFIIIA: 9 repeats; SP1: 3 repeats C4 zinc finger: 100 steroid hormone transcription factors Yang Xu, College of Life Sciences

9. Zinc finger binding with cis-element of DNA

10. Basic domain A basic domain is found in a number of DNA-binding proteins and is generally associated with: • the leucine zipper (ZIP) motif or • the helix-loop-helix (HLH) motif These are referred to as: • basic leucine zipper (bZIP) protein or • basic helix-loop-helix (bHLH) protein. Dimerization of the proteins brings together two basic domains which can then interact with DNA. Yang Xu, College of Life Sciences

11. C Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Zipper a-helix Basic domain N N Leucine zippers • contain a hydrophobic leucine residue at every seventh position. • is often at the C-terminal part of the bZIP protein. • These leucines are responsible for dimerization through inter-reaction between the a-helixes. • bZIP transcription factors contain Basic domainforms a clam around the DNA. Yang Xu, College of Life Sciences

12. C C N N DNA Helix-loop-helix Structure: Hydrophobic (憎水) residues on one side of the C-terminal a-helix allow dimerization. • a nonhelical loop of polypeptide chain separates twoα-helices in each monomeric protein. • HLH motif is often found adjacent to a basic domain that requires dimerization for DNA binding. Yang Xu, College of Life Sciences

13. a b Transcription activation domain-I Acidic activation domains: • have a very high proportion of acidic amino acids; • Trans-activation domains of yeast Gal4 (a) and mammalian glucocorticoid receptor (b); • are characteristic of many transcription activation domains. Yang Xu, College of Life Sciences

14. Transcription activation domain-II Glutamine-rich domains • have a very high proportion of glutamine amino acids; • In two activation regions of the transcription factor SP1 (TATA box). Yang Xu, College of Life Sciences

15. AP2 Transcription activation domain-III Proline-rich domain • a continuous run of proline residues can activate transcription; • For example, in the c-Jun, AP2 and Oct-2 transcription factors Yang Xu, College of Life Sciences

16. Inactivated Trans-factor Conformation Changed signal Activated Trans-factor Promote transcription binding DNA Transcription activation domain-IV • Transcription activation domain • have a very high proportion of acidic amino acid (also called acidic domain or acid blobs or negative noodles) Activation of Tans-factor Yang Xu, College of Life Sciences

17. N2 Examples of TranscriptionalRegulation • Constitutive transcription factors: SP1 • Hormonal regulation: steroid hormone receptors • Regulation by phosphorylation: STAT proteins • Transcription elongation: HIV Tat • Cell determination: myoD • Embryonic development: homeodomain proteins Yang Xu, College of Life Sciences

18. TAFII110 TFIID TAFII110 TBP TBP SP1 SP1 +1 GGGCGG TATA Housekeeping gene Promoter Constitutive transcription factors: SP1 SP1 is a very common transcription factor which contains (SP1 is present in all cell types): • three zinc finger motifs and • two glutamine-rich transactivation domains. General factors Yang Xu, College of Life Sciences

19. Inhibitor (HSP90) Hormonal regulation: steroid hormone receptors Steroid hormones are lipid soluble & can diffuse through cell membranes The TF called steroid hormone receptors. In the absence of the steroid hormone, the receptor is bound to an inhibitor, and located in the cytoplasm. The steroid hormone binds to the receptor and releases the receptor The receptor to dimerize and translocate to the nucleus. The DNA-binding domain of the steroid hormone receptor then interacts with its specific DNA-binding sites. Yang Xu, College of Life Sciences

20. Regulation by phosphorylation:STAT proteins Receptor JAK Signal transduction. This process often involves protein phosphorylation Interferon- induces phosphorylation of a transcription factor called STATIa through activation of the intracellular kinase called Janus activated kinase. When STATIa protein is unphosphoryl- ated, it exists as a monomer in the cell cytoplasm, when STATIa becomes phosphorylated at a specific tyrosine residue, it is able to form a homodimer which moves from the cytoplasm into the nucleus & bind to a DNA-binding. Yang Xu, College of Life Sciences

21. Trans-Initiation Complex Pol II Cellular factor Tat Transcription elongation: HIV Tat CTD CTD • Tat is protein encoded by HIV • Tat can make mammalian cells in transcription elongationstate. • Tat binds to an RNA stem-loop structure called TAR, just after the HIV transcription start site. TFIIH TAR • Tat binds to TAR on one transcript in a complex together with cellular RNA-binding factors. • This protein-RNA complex may result in the activation of the kinase activity of TFIIH. • As a result, the polymerase is able to read through the HIV transcription unit, leading to the productive synthesis of HIV proteins. • This leads to phosphorylation of the CTD of RNA Pol II, Yang Xu, College of Life Sciences

22. myoD gene P21 waf1/cip1 gene myoD protein P21 waf1/cip1 Cell determination: myoD myoD is a transcription factor. Cell determination Somites cell Muscle cell CDK Muscle cell Fibroblast Yang Xu, College of Life Sciences

23. Helix-turn-helix domain and binding with DNA 3 2 2 3 1 1 Embryonic development: homeodomain proteins • Homeodomain protein is a TF which was first found in Drosophila • It is encoded by homeobox,or homeotic gene inDrosophila Recognition domain • Homeotic genes are respon-sible for the correct specification of body parts. • For example, a gene mutation called antennapedia causes legs to grow where antennae would normally be p349 Fig. 12.11 Yang Xu, College of Life Sciences

24. That’s all for Section N Yang Xu, College of Life Sciences