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Eukaryotic Transcription II chapter 7

3. Eukaryotic Transcription II chapter 7. transcription initiation of some eukaryotic genes is mediated by a so-called initiator sequence, which is however not very well defined

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Eukaryotic Transcription II chapter 7

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  2. Eukaryotic Transcription IIchapter 7

  3. transcription initiation ofsome eukaryotic genesis mediated by a so-calledinitiator sequence,which is however not very well defined transcription of theoverwhelming majority of eukaryotic genesis mediated by a highly conservedregion termed theTATA box, whichserves to position and orient the RNA polymerase TATA sequence is very common in the promoters of protein coding genes

  4. TATA-box Binding Protein (TBP) The conserved C-terminal domain of TBP binds to the minor groove of the TATA box DNA, disrupting the normal helix structure and thereby bending the DNA dramatically. Curiously, although some promoters are “TATA-less”, they still require TBP for transcriptional initiation

  5. TBP is a component in all classes of eukaryotic transcription

  6. RNA polymerase II: it's complex! formation of a pre-initiation complex is the rate limiting step of transcription

  7. bacteriophage repressor 434 homodimer recognition helices Transcription factors recognize specific DNA sequence motifs Commonly,transcription factors possess an alpha-helical domain, the so-called RECOGNITION HELIX. The recognition helix can recognize a particular DNA sequence motif through non-covalent interactions with atoms in the bases. Recognition usuallyoccurs through interaction of the helix with the major groove of DNA.

  8. subregions of the promoter required for activation of transcription Transcription factor binding sites can be found through linker scanningmutations Reporter genes facilitate the relative quantification of transcriptional efficacy Some common “Reporters”: -Green Fluorescent Protein (GFP) --galactosidase (lacZ) -thymidine kinase (tk) -luciferase (luc) -chloramphenicol acetyltransferase (CAT)

  9. Electrophoretic mobility shift assays (EMSA) indicate general DNA binding activity • EMSA or gel/band/mobility shift assays are commonly used during purification of transcription factors to assay for DNA binding activity • A radiolabelled dsDNA segment that corresponds to a transcriptional regulatory region is used as a probe • If a polypeptide or protein complex present in a mixture can bind to the labelled DNA the probe’s mobility in the non-denaturing polyacrylamide gel will be retarded compared to free probe • EMSA cannot reveal the precise sequence that is bound by the protein!!!

  10. the samples are treated with randomly cutting DNAse I enzyme (partial digestion so roughly only one cut per labelled molecule) and the resulting DNA fragments are separated by gel electrophoresis column fractions of a cellular extract (which separates proteins based on various biochemical characteristics) are incubated with a radioactively labeled DNA fragment containing the transcription factor binding site Potential transcription factor binding region identified through linker scanning or gel shift assays DNA binding activity can be visualized in vitro region protected from DNAse I digestion by transcription factor binding

  11. most transcription factors have several domains that each perform distinct functions an example: the GAL4 transcription factor from yeast -> contains a DNA binding domain to bind UASGAL -> contains an activation domain to stimulate transcription Transcription factors have a modular structure GAL4

  12. transcription factors can contain domains for: DNA binding transcription activation transcription repression chromatin remodelling nuclear import protein interaction Modular structure is very common in eukaryotic transcription factors

  13. The Drosophila Antennapedia mutant wild type mutant • homeotic transformation: • for instance, mutation of the Drosophila gene Antennapedia leads to the formation of legs instead of antennae on a Drosophila head segment

  14. The homeodomain was named due to its presence in several transcription factors that give rise to homeotic transformations when mutated at particular residues Homeodomain proteins homeodomain binding sites

  15. C2H2 types usually contain three or more finger units and bind to DNA as monomers C4 types usually contain only two finger units and bind to DNA as homo- or heterodimers ie…steroid hormone receptors the C6 Zinc Finger transcription factor is a variation wherein six cysteine metal ligands coordinately bind two Zn2+ ions Different types of zinc finger DNA binding domains exist

  16. Leucine zipper proteins • leucine zipper proteins bind DNA exclusively as homo- or heterodimerswith their extended alpha-helices, which bind the DNA’s major groove • they contain a leucine or a different hydrophobic amino acid in every seventh position in the C-terminal region of the DNA binding domain • these hydrophobic residues form a coiled coil domain, which is required for dimerization

  17. Helix-Loop-Helix proteins • helix-loop-helix (HLH) proteins are very similar to leucine zipper proteins,however instead of an extended alpha-helix they are characterized by two alpha-helices, which are connected by a short loop • HLH proteins contain hydrophobic amino acids spaced at intervals characteristic of an amphipathic alpha-helix in the C-terminal region of the DNA binding domain

  18. protein-protein interaction favours formation of, and stabilises the ternary complex (DNA Binding) Transcription factors of unrelated classes can bind COOPERATIVELY SOME TRANSCRIPTION FACTORS ACTUALLY REQUIRE COACTIVATORS…

  19. The combination of transcription factor binding sites in promoters, paired with the potential for protein-protein interactions among the transcription factors to enhance the diversity of transcriptional responses Homo- and heterodimer formation is common among transcription factors, ie…three transcription factors that can homodimerize or heterodimerize=> 6 different possible combinations Combinatorial possibilities greatly extend the potential for diversified gene regulation

  20. Arabidopsis flowers consist of concentric whorls of organs: four sepals in the outer whorl surround four petals in the next whorl, followed by six stamens and two carpels. These organs are specified by the combinatorial action of a limited number of related transcription factors of the so called MADS box class. An example: specification of floral organs in Arabidopsis

  21. MADS box transcription factors involved in floral organ formation are expressed in distinct patterns. The expression patterns lead to the formation of distinct multimeric MADs box factor complexes in the different whorls. The different MADS box complexes have different affinities for MADS box target DNA sequences, thus establishing differential expression of target genes. MADS box factors specifying flower organs are expressed in specific domains This is best described by an “ABC” model to explain floral organ patterning!

  22. The MADS box factors in floral identity: “A” function:APETALA1 “B” function:APETALA3 PISTILLATA “C” function:AGAMOUS An important accessory factor: SEPALLATA 3 note: only AP1 and SEP3 contain transcription activation domains!!! sepal petal stamen carpel organ domain: AP3 MADS box factor expression: PI AP1 AG SEP3 MADS box factor complex: AP1 AP1 PI AP3 PI AP3 AG SEP 3 AP1 SEP 3 AG SEP 3

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