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Section N Regulation of Transcription in Eukaryotes N1 Eukaryotic Transcription Factors N2 Examples of Transcriptional Regulation Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences 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 Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences Structure of a typical eukaryotic gene Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences 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 DNA-binding domains Consist of: • Helix-turn-Helix: (a 60aa homeodomain) • Zinc finger domain: (C2H2 and C4 zinc finger) • Basic domain: (bZIP or bHLH) Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences Helix-turn-helix domain Structure: a 60aa homeodomain encoded by the homeobox. Found in: Helix • Antennapedia TF of Dropphila • Phage DNA-binding proteins such as the l cro repressor; Turn Recognition helix • Lac and trp repressors; • cAMP receptor protein, CRP. DNA-binding domain: • Recognition helix, lies partly in the major groove and interacts with the DNA. Section N: Regulation of transcrip. in Euk. DNA Yang Xu, College of Life Sciences Helix-turn-helix domain and binding with DNA Recognition helix 2 2 3 3 1 1 Zinc finger domain-I DNA binding sites R C N C Zn R H H R H C H Zn C H Peptide chain C2H2 zinc finger: C TFIIIA: 9 repeats; SP1: 3 repeats C4 zinc finger: C N C Zn C C C C Zn 100 steroid hormone transcription factors C C Section N: Regulation of transcrip. in Euk. C Yang Xu, College of Life Sciences Zinc finger binding with cis-element of DNA Basic domain A basic domain is found in a number of DNAbinding 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. Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences Leucine zippers • contain a hydrophobic leucine residue at every seventh position. • is often at the C-terminal part of the bZIP protein. C Leu Zipper N Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu a-helix Basic domain Section N: Regulation of transcrip. in Euk. N • These leucines are responsible for dimerization through interreaction between the a-helixes. • bZIP transcription factors contain Basic domain forms a clam around the DNA. Yang Xu, College of Life Sciences Helix-loop-helix C Structure: Hydrophobic (憎水) residues on one side of the C-terminal a-helix allow dimerization. • a nonhelical loop of polypeptide chain separates two α-helices in each monomeric protein. • HLH motif is often found adjacent to a basic domain that requires dimerization for DNA binding. C N N DNA Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences Transcription activation domain-I Acidic activation domains: a • 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. b Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences 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). Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences Transcription activation domain-III Proline-rich domain AP2 • a continuous run of proline residues can activate transcription; • For example, in the c-Jun, AP2 and Oct-2 transcription factors Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences 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 Inactivated Trans-factor Conformation Changed signal Activated Trans-factor Promote transcription binding DNA Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences N2 Examples of Transcriptional Regulation • • • • • • 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 Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences 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. TAFII110 SP1 TAFII110 TBP SP1 GGGCGG TATA TBP TFIID General factors +1 Housekeeping gene Promoter Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences Hormonal regulation: steroid hormone receptors Inhibitor (HSP90) 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. Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences Regulation by phosphorylation: STAT proteins 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. Receptor JAK When STATIa protein is unphosphorylated, 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. Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences Transcription elongation: HIV Tat • Tat is protein encoded by HIV • Tat can make mammalian cells in transcription elongation state. • Tat binds to an RNA stem-loop structure called TAR, just after the HIV transcription start site. Trans-Initiation Complex TFIIH Pol II CTD 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. • This leads to phosphorylation of the CTD of RNA Pol II, Section N: Regulation of transcrip. in Euk. Cellular factor • As a result, the polymerase is able to read through the HIV transcription unit, leading to the productive synthesis of HIV proteins. Tat Yang Xu, College of Life Sciences Cell determination: myoD myoD is a transcription factor. Somites cell Cell determination myoD gene myoD protein Section N: Regulation of transcrip. in Euk. Muscle cell P21 waf1/cip1 gene P21 waf1/cip1 CDK Muscle cell Fibroblast Yang Xu, College of Life Sciences Embryonic development: homeodomain proteins • Homeodomain protein is a TF which was first found in Drosophila Helix-turn-helix domain and binding with DNA 3 2 2 3 1 1 • It is encoded by homeobox, or homeotic gene in Drosophila Recognition domain • Homeotic genes are responsible for the correct specification of body parts. • For example, a gene mutation called antennapedia causes legs to grow where antennae would normally be Section N: Regulation of transcrip. in Euk. p349 Fig. 12.11 Yang Xu, College of Life Sciences That’s all for Section N Section N: Regulation of transcrip. in Euk. Yang Xu, College of Life Sciences