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Regulation of TranscriptionI. Basal vs. activated transcription for
mRNA genesA. General transcription factor (TF) vs. promoter-specific
1. general TFs are required by all mRNA genesa. an absolute requirementb. transcription can occur alone with these
factors is by definition the basal level of transcription2. promoter-specific TFs are different for each gene3. 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
Targets of Activation Domains
• General Transcription Factors– TBP (TFIID)– TFIIB– TFIIA
• RNA polymerase II• Coactivators
– Mediator– TFIID
Role of Activation Domains
1. Recruitment• Bind Faster• Remain bound longer
2. Conformational change• Active vs. inactive configuration• Blocked vs. accessible
3. Covalent modification
Regulation of TranscriptionII. Question of Activation
A. diversity of response - combinatorial effect1. properties of response elements (RE)2. relatedness of RE and enhancers3. trans acting factors
induction: heat shock, heavy metals, viral infection, growth factors, steroids
4. greater multiplicity with combinatorial approachB. Master gene regulatory proteins
1. response elements shared2. example of homeodomains
Regulation of TranscriptionII. Question of Activation
A. diversity of response - combinatorial effect1. properties of response elements (RE)2. relatedness of RE and enhancers3. trans acting factors
induction: heat shock, heavy metals, viral infection, growth factors, steroids
4. greater multiplicity with combinatorial approachB. Master gene regulatory proteins
1. response elements shared2. example of homeodomains
C. regulating the activity of the transcription factors
DNA binding domainsA. Zinc fingers
1. Cys2-His2 fingers: Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X-Hisa. example is TFIIIA has 9 Zn finger repeatb. typically the number of fingers range from 2-9c. can be involved in binding to RNAd. not all Zn fingers are used to bind DNA, nor are they always
part of a transcription factor2. Cys2-Cys2 fingers: Cys-X2-Cys-X13-Cys-X2-Cys
a. found in steroid receptorsb. typically nonrepetitivec. binding sites are short palindromesd. bind as dimers
3. Binuclear Cys6 finger: Gal4 DNA binding domains
DNA binding domainsB. Steroid receptors
1. Ligand mediated activation2. Functional Domains
a. DNA bindingb. ligand binding - hormonec. activation domain
3. Two classesa. form homodimers: bind consensus half site (TGTTCT, except for ER is TGACCT)b. form heterodimers: bind half sites of
TGACCT, direct repeatsc. spacing of the half sites is crucial for the
degree of specificity
DNA binding domainsC. Leucine zippers - dimer formation
1. brings 2 DNA binding domainsin close juxtaposition
example is Gal4 2. amphipathic alpha helices with
Leu residues on one faceLeu repeats every 7 amino acid
3. interface forms a coiled coil
DNA binding domainsD. bZIP example is GCN5
1. basic region attached to a leucine zipper2. is a dimer kept together by the leucine
zipper3. an alpha helic containing basic residues
contacts the major groove of DNA4. contacts are made with the portion of the
bases exposed in the major groove and some phosphate backbone contacts
DNA binding domains
E. bHLH domain1. basic helix loop helix motif2. positively charged alpha helix binds
to major groove3. two other alpha helices form a four
helix bundle in dimer4. many will also contain a leucine
zipper
Activation Domains
A. Acidic activators - example of Gal4pB. Glutamine rich domainC. Proline rich domain
Transcription ElongationA. General
1. in vivo rates are 1200-2000 nucleotides/min
2. in vitro rates are 100-300 nucleotides/min3. elongation is not a monotonic continuous
processa. there are strong pause sitesb. effects of chromatin on process
4. pausing versus arrest (definition of)
Transcription ElongationB. Negative elongation factors (N-TEFs)
1. DSIF2. factor 2
C. Positive elongation factors (P-TEFs)1. prevent sequence dependent arrest (i.e. TFIIS or SII)
nucleolytic cleavage/ backtracking2. catalytic activity (TFIIF, elongin, ELL complex)3. regulates the rate of elongation
through chromatin (FACT)