TFs and their study

Question 1

DNA-binding domain (DBD)

Answer 1

Part of TFs that mediates direct interaction with the DNA, at the level of a specific recognition sequence (motif)

Question 2

Main classes of DNA-binding domains

Answer 2

• Helix-turn-helix (HTH)
• Zinc finger (ZNF)

Question 3

Helix-turn-helix DBD

Answer 3

in both prokaryotes and eukaryotes
2x α-helices, seperated by ß- turn
ß-turn allows orienting the second α-helix (recognition helix) in a way that allows it to fit inside the major groove.

Question 4

Homeodomain

Answer 4

Special case of HTH found in eukaryotes
3-4x α-helices, with helicases 2 and 3 separated by a ß-turn
Helix 3 acts as the recognition helix
Helix 1 stabilizes the structure

Question 5

Zinc-finger DBD

Answer 5

• Mostly in eukaryotes
• Most common -> Cys2His2 finger
• The α-helix contacts the DNA at major groove
• The ß-sheet ineracts with suger-phosphate backbone
• zinc atom essential for correct orientation of the 2 fingers

Question 6

In vitro approaches for studying DNA-TF interactions

Answer 6

1. Gel retardation / gel shift
2. Protection assay

Question 7

In vivo method for studying DNA-TF interactions

Answer 7

Chromatin immunoprecipitation

Question 8

Gel retardation

Answer 8

• Stable binding of TF to specific DNA sequence
• Sample with only DNA and sample with both DNA and TF are loaded on the gel
• When TF is bound to a piece of DNA it will be heavier and move more slowly when compaired to only DNA

Question 9

Protection Assay DNase I approach

Answer 9

Based on assumption that the portion of DNA that is bound by TF, will be protected from modification.
- DNA is digested with DNase I, under limiting conditions.
- When there is no TF -> DNase I will cut everywhere ( the limiting conditions make sure it’s only 1 cut per DNA molecule)
- WHen TF is bound DNase I cuts everywhere except at the place of binding -> place where TF is bound is protected from cutting.
- Results in an empty region in the banding pattern. (footprint)

Question 10

DMS footprinting

Answer 10

• DNA is treated with DMS ( methylates N7 of G)
• Piperidine then leads to DNA strand breaks at DMS-modified positions
• TF bund bases are protected form DMA methylation, leaving an empty region in the bnding pattern -> footprint.

Question 11

Chromatin immunoprecipitation
(what is it?)

Answer 11

• Chromatin immunoprecipitaton (ChIP) allows study of DNA protein intractions in living cells
• Sites of binding of the assayed TF are identified troughout the entire genome, in 1 experiment
• Uses antibodies to specifically recognize the TF of interest and enrich it from cellular extracts,along with the interacting DNA.

Question 12

Chromatin immunorecipitation steps

Answer 12

• To stabalize DNA-TF interactions, cells are treated with chemical reagents (formaldehyde) -> formation of covalent crosslinks between protein and nucleic acids
• Cells are lysed an genome is randomly fragmented by sonification
• Antibody targeted to the TF to recover TF-bound DNA fragments
• Accomplished by magnetic beads conjugated to protein G
• Crosslink reversal by temperature
• Released DNA is sequenced
• Map each fragment back to its original position on genome
• Genome-wide map of TF binding sites