RR5

Question 1

What is EMSA?

Answer 1

Combine DNA with protein mixture - run on agarose gel
Whichever proteins bind to DNA fragments will not move down the gel as fast/far

Question 2

Describe TF assay testing using plasmid cotransfection. (This would be after protein identification by EMSA)

Answer 2

Testing it for availibility to activate transcription
Transfect two expression vectors:
1- cDNA of the identified protein
2- Regulatory site recognized by protein with reporter gene (lacZ, GFP…)

-Transfect both vectors into cell, if cDNA makes protein, it will be able to recognize its control elements - see if cloned protein can activate transcription

Question 3

What are recognition helices?

Answer 3

Alpha helices rich in positive amino acids allow for positive interactions with major DNA grooves

Question 4

Modularity of transcription factors?

Answer 4

Transcription factors have multiple domains that each perform specific functions
- They interact with specific sequences, either enhancing or repressing transcription

Question 5

What is function of GAL4?

Answer 5

• Binds to UASgal and will activate transcription of genes required to metabolize galactose

Question 6

What is needed for transcription to start?

Answer 6

• At least 2 different domains
• N region (required for binding)

Question 7

What are 6 types of domains TFs can possess?

Answer 7

• DNA binding
• Transcription activation
• Transcription repression
• Chromatin remodelling
• Nuclear import
• Protein interaction

Question 8

What are homeodomain proteins?

Answer 8

Present in several transcription factors that give rise to homeotic transformations when mutated (body formation)

Question 9

What are 3 types of zinc fingers? Do they bind as monomers?

Answer 9

Structure formed when zinc links with transcription factors
- C2H2 - 2 cysteine, 2 hysteideines, binds to DNA as monomers
- C4 - 2 units, bind to homo or heterodimers (4 cysteines)
- C6 - 6 cysteine binds to two ZN2+ ions, bind as monomers

Question 10

What is a leucine zipper protein?

Answer 10

Acts as a hydrophobic region encouraging dimer formation
- Positions helices so they recognize DNA target
- Contain leucine every 7th position in the C-terminal region, forming a coiled coil

Question 11

What is crucial to protein dimer formation?

Answer 11

hydrophobic interfaces - critical for protein positioning

Question 12

What is a helix loop helix protein?

Answer 12

Characterized by 2 alpha helices connected by a short loop
- contain hydrophobic a.a’s spaced at intervals characteristic of amphipathic alpha-helix in C terminal region of DNA binding domain

Question 13

How might we see difference between HLH and leucine zipper?

Answer 13

Can see the loop formed by HLH

Question 14

What is cooperative DNA binding? Explain Ap1 + NFAT example

Answer 14

Combination of possibilities expands potential for diversified gene regulation

AP1 and NFAT - don’t interact strongly with binding site - together have big increase in ability to bind to DNA

protein/protein interactions normally stabilize

Question 15

Can TFs bind coopertively? How many possibilities could this yield?

Answer 15

Yes!
3 TFs that can homo or heterodimerize yields 6 possible combos

Question 16

Explain cHIP

Answer 16

• Crosslink macromolecules with formaldehyde
• Shear DNA into small fragments
• Immunoprecipitate with protein/antibody - the protein:fragment complexes stay in column <- this is our cHIP

Question 17

What is the mediator?

Answer 17

• “multi subunit giant protein”
• Bridges vast sections of chromatin to enhance transcriptional initiation
• Allows positioning of RNA pol II close to gene it wants to effect
• Transcription occurs in “loop like” manner, the mediator ensures the loop doesn’t pull apart