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32 Cards in this Set
- Front
- Back
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What is UAS? |
Upstream Activating Sequence |
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What is the function of UAS? |
UAS bind upstream of the basal promoter to give high levels of transcription with the basal promoter and therefore high levels of proteins produced. |
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What are the 2 classes of UAS? |
'Common' sequence elements Response elements |
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What are 'common' sequence elements? |
Often located close to the core promoter. Bind activators that are relatively abundant and constitutively active (will always activate transcription). |
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What are examples of 'common' sequence elements? |
GC box (GGGCGG) - factor Sp1 Octamer (ATTTGCAT) - factor Oct-1 CAAT box (GGCCAATCT) - factor NF1 |
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What are examples of response elements? |
1) SRE (TGACTCA) Binds: Serum response factor (responds to growth factors in the medium) Inducer: Serum 2) HSE (CTNGAATNTTCTAGA) Binds: Heat shock factor Inducer: Heat shock (in presence of heat shock, transcription is activated. Without, it isn't) |
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What are response elements? |
Bind factors whose activity is controlled (induced) in response to specific stimuli. |
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What is combinatorial control of transcription? |
Combination of elements dictates when and at what level a gene is transcribed. (look at diagram) |
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Does enhancer location affect their ability to work? |
No, enhancers work irrespective of where the are located. They will still activate if located upstream, downstream or if the orientation of the binding site is switched. |
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How do enhancers still work if they are very far apart? |
Can loop round so the binding-site machinery and basal transcription machinery have contact. This works as DNA is flexible so it doesn't matter where the activator binds. |
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What domains do eukaryotic activators contain? |
They are modular structures separated by flexible domains. Usually contain one DNA binding domain but can have multiple activation domains. |
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What are examples of DNA binding domains? |
Leucine zipper Zinc finger Homeodomain Helix loop Helix |
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What activation domains usually characterised by? |
Often characterised according to their amino acid composition (lack of sequence conservation and structural info). Contain multiple short segments that work together in an additive fashion (sticky bits of protein). Eg/// Acidic patch (VP16) - clusters of negatively charged residues (rich in acidic residues (asp/glu)) Glutamine rich (SP1) - high glen content Proline rich (Jun) - v. important human transcription factor. |
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What is an in vivo method for analysis of activators? |
Reporter assays |
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What are in vitro methods for the analysis of activators? |
DNA footprinting Electrophoretic Mobility Shift Assays (Gel Shift - ESMA) Transcription assays |
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What do reporter assays do? |
Measure activator function - how well it binds to DNA to activate transcription. |
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What is the process of reporter assays? |
Plasmid 1 = plasmid expressing activator of interest. Plasmid 2 = plasmid containing the reporter gene and upstream of the basal promoter there is a binding site for the activator of interest. 1) both plasmids are put into/transfected into the cell (nucleus). 2) the transcriptional activator is expressed from plasmid 1 (protein). 3) protein is made and translocated to the nucleus. 4) the activator protein binds to the binding site on plasmid 2. 5) this activates transcription. 6) the expression of the reporter gene is switched on. 7) this produces reporter-gene transcripts and reporter enzyme. 8) the activity of reporter gives a read-out of the activity of the transcriptional activator. |
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What does EMSA/gel shift do? |
Measures the binding ability of the activator (ability of activator to bind DNA). |
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How is EMSA carried out? |
1) mix purified activator and radio labelled probe DNA (short ds-oligonucleotide labelled with p32). 2) run this mixture on a non-denaturing acrylamide gel. The free DNA is a lot smaller than the protein-DNA complex therefore runs fast down the gel (free DNA = DNA from the probe that didn't bind to the activator). The DNA bound with a protein is a lot bigger so will migrate through the acrylamide gel slower. Gel shift = activator that did bind moves slower. |
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What does a transcriptional assay do? |
Measures the transcription ability of an activator. |
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How is a transcription assay carried out? |
Put in all the bits you need to reconstitute transcription: RNA pol ll, GTFs, DNA template, radiolabelled rNTPs In the absence of activator = very low levels of RNA transcripts. With activator = radiolabelled transcripts show up as bands. |
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What do functional assays require? |
Require the activator to have both a fictional DNA binding domain and a functional activation domain. Using ESMA and Transcription assay. (look at diagram) |
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What is DNA microarray analysis? |
Looking over all the genes to see which one the transcription factor effects. Compares the relative amounts in the presence and absence of the TF. |
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If a red fluorescent label is the control cells (where activator is present) and a green fluorescent label is the cells lacking the transcription factor, what do the different coloured spots represent when hybridised to a microarray? |
RED = genes decreased in the absence of transcription factor (as there is more RNA in the control cell than there is in the cells lacking TF) - could be potential activator binding targets. GREEN = genes increased in the absence of the transcription factor (as there is more RNA in the calls lacking the TF than there is in the control cells) - likely to be a repressor. YELLOW = genes whose expression level does not change (expression is the same in both the control cells and cells lacking TF). BLACK = no RNA present. |
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What is chromatin immunoprecipitation (ChIP)? |
Tells you where a particular protein is binding in the genome (can look at whole genome). |
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How is chromatin immunoprecipitation carried out? |
1) use chemical cross-linking agent (formaldehyde) which will cross-link DNA-binding proteins to DNA) - activator binding to binding site. 2) isolate DNA (chromatin) and shear the DNA up into small pieces. 3) then use an antibody to the protein you're interested in to enrich and purify only the bits of DNA that are bound to the activator and remove everything else (precipitate chromatin with protein-specific antibody). 4) use heat to reverse the cross link (get rid of protein so just have activator binding sites). 5) analyse the DNA using: i) PCR ii) microarray (ChIP on CHIP) iii) sequencing (ChiP-seq) |
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What are the 3 ways that activators work by? |
1) Promote binding of an additional factor - the binding of one activator can promote/stimulate the binding of an extra activator. 2) Stimulate complex assembly (recruitment) 3) Release stalled RNA polymerase (stimulate activity) |
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Do activators work synergistically? |
Yes, they are more than just additive. |
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What components of the PIC complex do activators interact with to promote assembly? |
TFIID (via TAFs) TFIIB Mediator Activators can increase the rate of PIC formation by: - increasing TFIID binding - increasing TFIIB binding - increasing RNA pol ll recruitment. |
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What is mediator? |
Mediator is the link between the activator and basal transcription machinery. It is a huge complex (22 polypeptides). It can exist on its own or associated with RNA pol ll - through the CTD. Composed of 3 domains: head, middle and tail |
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What is the function of mediator? |
Mediator provides a bridge between activators and RNA pol ll. This aids the recruitment of RNA pol ll and therefore enhance PIC formation/assembly. |
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What is an example of active activator proteins that can release the stalled RNA pol ll? |
Heat shock genes such as hsp70. |
