What is the difference between basal and activated transcription?
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1 What is the difference between basal and activated transcription?
2 Regulation of Transcription I. Basal vs. activated transcription for mrna genes A. General transcription factor (TF) vs. promoterspecific 1. general TFs are required by all mrna genes a. an absolute requirement b. transcription can occur alone with these factors is by definition the basal level of transcription 2. promoter-specific TFs are different for each gene 3. the promoter-specific TFs are required for maximal level of transcription or for activated transcription (induction) B. a third state is that of a repressed state
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4 Regulation of Transcription What is the role of the response elements and associated factors
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7 How Can You Test the Functional Activity of an Activation Domain?
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9 Targets of Activation Domains General Transcription Factors TBP (TFIID) TFIIB TFIIA RNA polymerase II Coactivators Mediator TFIID
10 Role of Activation Domains 1. Recruitment Bind Faster Remain bound longer 2. Conformational change Active vs. inactive configuration Blocked vs. accessible 3. Covalent modification
11 Coactivator versus Repressor (corepressor) Activation and Repression of Transcription
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15 Regulation of Transcription II. Question of Activation A. diversity of response - combinatorial effect 1. properties of response elements (RE) 2. relatedness of RE and enhancers 3. trans acting factors induction: heat shock, heavy metals, viral infection, growth factors, steroids 4. greater multiplicity with combinatorial approach B. Master gene regulatory proteins 1. response elements shared 2. example of homeodomains
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17 Regulation of Transcription II. Question of Activation A. diversity of response - combinatorial effect 1. properties of response elements (RE) 2. relatedness of RE and enhancers 3. trans acting factors induction: heat shock, heavy metals, viral infection, growth factors, steroids 4. greater multiplicity with combinatorial approach B. Master gene regulatory proteins 1. response elements shared 2. example of homeodomains C. regulating the activity of the transcription factors
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20 What are the DNA binding domains of the TFs?
21 DNA binding domains A. Zinc fingers 1. Cys2-His2 fingers: Cys-X 2-4 -Cys-X 3 -Phe-X 5 -Leu-X 2 -His-X-His a. example is TFIIIA has 9 Zn finger repeat b. typically the number of fingers range from 2-9 c. can be involved in binding to RNA d. not all Zn fingers are used to bind DNA, nor are they always part of a transcription factor 2. Cys2-Cys2 fingers: Cys-X 2 -Cys-X 13 -Cys-X 2 -Cys a. found in steroid receptors b. typically nonrepetitive c. binding sites are short palindromes d. bind as dimers 3. Binuclear Cys6 finger: Gal4 DNA binding domains
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26 DNA binding domains B. Steroid receptors
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30 DNA binding domains B. Steroid receptors 1. Ligand mediated activation 2. Functional Domains a. DNA binding b. ligand binding - hormone c. activation domain 3. Two classes a. form homodimers: bind consensus half site (TGTTCT, except for ER is TGACCT) b. form heterodimers: bind half sites of TGACCT, direct repeats c. spacing of the half sites is crucial for the degree of specificity
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34 DNA binding domains C. Leucine zippers - dimer formation 1. brings 2 DNA binding domains in close juxtaposition example is Gal4 2. amphipathic alpha helices with Leu residues on one face Leu repeats every 7 amino acid 3. interface forms a coiled coil
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40 DNA binding domains D. bzip example is GCN5 1. basic region attached to a leucine zipper 2. is a dimer kept together by the leucine zipper 3. an alpha helic containing basic residues contacts the major groove of DNA 4. contacts are made with the portion of the bases exposed in the major groove and some phosphate backbone contacts
41 DNA binding domains E. bhlh domain 1. basic helix loop helix motif 2. positively charged alpha helix binds to major groove 3. two other alpha helices form a four helix bundle in dimer 4. many will also contain a leucine zipper
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44 DNA binding domains Dimer formation regulates the activity of the transcription factor
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46 Activation Domains A. Acidic activators - example of Gal4p B. Glutamine rich domain C. Proline rich domain
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49 Transcription Elongation A. General 1. in vivo rates are nucleotides/min 2. in vitro rates are nucleotides/min 3. elongation is not a monotonic continuous process a. there are strong pause sites b. effects of chromatin on process 4. pausing versus arrest (definition of)
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51 Transcription Elongation B. Negative elongation factors (N-TEFs) 1. DSIF 2. factor 2 C. Positive elongation factors (P-TEFs) 1. prevent sequence dependent arrest (i.e. TFIIS or SII) nucleolytic cleavage/ backtracking 2. catalytic activity (TFIIF, elongin, ELL complex) 3. regulates the rate of elongation through chromatin (FACT)
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