RR5 Flashcards
How can regulatory sites be found and what can be identified?
They can be found through linker-scanning mutations. They can identify reporter genes such as GFP, B galactosidase, Thymidine kinase, Luciferase, Chloramphenicol acetyltransferase.
What is EMSA? (Electrophoretic Mobility Shift Assays)
It is a method to detect a given protein or complex that can interact with a specific segment of DNA, which forms a protein-DNA complex with its own inherent proteins. It can label segments of interest.
Make probes that are radio-labelled or fluorescently labelled, that correspond to double stranded DNA sequence.
- Take labelled segment and combine it with nuclear extract which forms the protein-DNA complex.
- The nuclear extract is used as a starting material, then it is separated using column chromatography, each fraction is a different protein complement.
- Something in the nuclear extract will bind to radio labelled DNA< will identify complexes that form.
How would you test protein ability to activate transcription in vivo? (in a living organism)
1) Generate cDNA of protein and put it in an expression vector
2) have another vector that contains cis-regulatory site that protein uses to enhance transcription
3) transfect both vectors into cell, if cDNA makes protein, it will be able to recognize its control elements -> activate transcription of reporter gene
How do transcription factors recognize specific motifs?
They interact with specific sequences, either enhancing or repressing transcription.
There are recognition helices: alpha helices rich in positive amino acids that allow for interactions with major DNA grooves.
Why do transcription factors have multiple domains?
They each perform different functions
How many regions do you need to activate transcription?
At least two regions
What is the function of Gal4 transcription factor yeast?
It binds to UASgal and activates transcription of genes required to metabolize galactose.
What are some things that transcription factors can possess domains for?
Dna binding
Transcription activation and repression
Chromatin remodelling
Nuclear import
Protein interaction
Will any transcriptional activation function activate transcription of downstream gene?
Yes
What are homeodomain proteins?
They are present in several transcription factors that give rise to homeotic (one body part is replaced by another) transformations when mutated.
What are Zinc Finger transcription factors and what are three types?
- Zinc finger transcription factors have structure formed when zinc links with TF- looks like a finger.
1) C2H2 - 2 cysteine, 2 hystideines bind to DNA as monomers
2) C4 - 2 units, bind as homo or heterodimers. 4 cysteines: nuclear receptors.
3) C6- 6 cysteine bind to two Zn2+ ions, bind as monomers
What are leucine zipper proteins?
They act as hydrophobic regions encouraging dimer formation. They position helices so they recognize DNA target. They contain leucine every 7th position int he C-terminal region, forming coiled coil. The hydrophobic interfaces are crucial to protein positioning.
What are helix-loop-helix proteins?
They are characterized by 2 alpha helices connected by a short loop. T
hey contain hydrophobic amino acids spaced at intervals characteristic of ampipathic a-helic in C terminal region of DNA binding domain.
What is an example of transcription factors binding cooperatively? Why would they want to do this?
An example is API and NFAT. Neither interact strong with the binding site but together they have a big increase in ability to bind to DNA.
What method should I use if I what DNA sequences transcription factors bind? How would this work?
Use chIP!!!
1) Crosslink macromolecules with formaldehyde
2) Shear DNA into small fragments
3) Immunoprecipitate with antibody which creates chIP
4) Use PCR and sequencing to find DNA bound by material by TFs
