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29 Cards in this Set
- Front
- Back
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TATA box is how many bp upstream? what is it it similar to in bacteria? |
~25 upstream, similar to -10 in bacteria |
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what is the Inr? |
it's the initiator located at +1, first mRNA base tends to be A in py2capy5 |
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what is the basal transcription apparatus? |
its the general factors TFII-whatever, plus RNAP II |
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what is the core promotoer? |
Downstream elements, TFIIB recognition (BRE), TATA box and lnr |
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what does the TBP do? what is this similar to? what TFII is it apart of? |
its apart of TFIID, TBP is the TATA binding protein and it binds the TATA box, it causes it to bend/distort just like CAP-camp to allow other TFs to bind |
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what is the structure of TFIID? |
TBP + 11 TAFs |
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what is the order assembly from RNAP II initiation complex? |
TFIID assembly (TBP and TAFs) to TFIIA to TFIIB to TFIIF to RNAP II to TFIIE to TFIIH to promoter clearance |
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what does TFIIA do? |
increases protection upstream and stabilizes TBP-TATA box interaction |
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what does TFIIB do? |
protection downstream of TATA box, binds BRE, helps position active site of RNAP II |
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what does TFIIF do? what are its subunits? |
it has 2 subunits, RAP 74(larger): which is ATP dependent helicase, RAP38 (smaller): has homology to bacterial sigma factor and regions that contact core polymerase thus binding tightly to RNAP II, it recruits RNAP II into the assembly complex |
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what accounts for RNAPII larger size? what is the DAB complex? |
DABPoIF complex: A,D,B,F + PolII DAB complex is just TFIIA, TFIID and TFIIB |
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what TF is responsible for promoter melting? how does it do this? what other activity does it have? |
TFIIH, it has a subunit that is like an ATP driven translocator of dsDNA and binds downstream of polymerase + feeds dsDNA w/ right handed threading into the cleft of the polymerase = action of melting DNA it has DNA helicase activity |
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describe TFIIH structure |
9 subunits, has 2 complexes: protein kinase complex, and a core TFIIH complex with 2 DNA helicase/atpase activity
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what are the two major roles of TFIIH in transcription initiation? |
1) to phosphorylate the CTD of RNA pol II 2) and unwind DNA at the transcription start site |
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what does TFIIE do? |
it extends protection region to +30 and recruits TFIIH |
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what kinase gets recruited by transcriptional activators to phosphorylate serine at position 2 to promote elongation? |
P-TEFb kinase |
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where do mutations that decrease transcription the most occur? |
TATA box, CAAT box (~75), GC box (~90) |
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what binds GC boxes? |
sp1 |
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what virus was used in experiments to show reduced transcription levels as GC box content declined? |
SV40 |
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how can you study promoters? what is this similar to? |
linker scanning mutagenesis, similar to yeast ARS |
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what happens when you do not have a TATA box? |
TBP tethers itself to TAFs bound at Inr |
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what happens when you do not have a TATA box but you have a GC box? |
TBP tethers itself to sp1 which is bound to GC boxes |
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what was H-chain enhancers used to show? |
that enhancers have tissue specificity, as well as enhancers reinserted into Ig gene in a different location, had no change in expression |
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how can enhancers interact with promoters on seperate DNAs? whats an example? |
NtrC activates RNAP by looping |
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what is the purpose of enhancer traps? |
enhancer trapping allows one to go aftergenes that may participate in the development of a particular tissue |
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what do most enhancer traps have? |
a transposable element (p element) and a reporter gene |
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what reporter gene did they have in the enhancer traps with drosophila? |
lac z gene reporter, it produces beta-galactosidase which can be targeted by an antibody, or blue staining |
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if an enhancer trap does not have a reporter gene such as GAL 4, what can you do? |
you can cross it with a reporter gene such as UAS, gal4 binds UAS. |
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how do you clone an enhancer and flanking DNA? |
first digest target DNA with restriction enzyme that will not cut reporter gene but will cut flanking DNA, recircularize the DNA by ligations and do PCR using primers based on the reporter gene that face opposite directions then inverse PCR amplifies joined flanking genomic sequences that can be sequenced |
