Domain Swaps in Molecular Genetics Flashcards Preview

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Flashcards in Domain Swaps in Molecular Genetics Deck (21):
1

Domains

- dimerization
- activation
- DNA binding

2

Domains are Distinct

- allows us to swap domains and carry out experiments to identify and study the functions of the separate domains.
- located in different parts of the protein and can act independently

3

LexA gene

- repression

4

GAL4 gene

- activation

5

lexA/GAL4

- fusion protein
- DBD - lexA
- activation/dimerization - GAL4

6

Recombinant protein

They are derived by procedures involving recombinant DNA.

7

Enhancers

- contain multiple binding sites for transcription factors
- binds activator
- GAL4 doesn't work well unless hooked up to an enhancer and enhancer-binding protein

8

regulatory region of genes

- where transcription factors interact with genes

9

upstream activating sequence

- DNA binding domain interacts with

10

transcription factor

- diffusible protein made elsewhere.
- most transcription factors bind as heterodimers

11

dimerization domain

- will bring two GAL4 together (dimerize)

12

activation and dimerization domain

- amino acids in these are covalently linked

13

DBD and DNA

- interactions between these two are non-covalent.

14

Silencers

- bound by repressors
- bind to lexO
- DNA sequences bound by lexA differ from sequences within upstream activating sequence bound by GAL4

15

Domain swap

- switch DBD of lexA with GAL4

16

Test using reporter genes

- LacZ gene produces B-galactosidase (easily measured)
- remove enhancers and silencers from endogenous location with genome and fuse to reporter gene that you can easily assay
- LacZ fused to UAS or LexO operator

17

GAL4

- activates transcription by binding to UAS in enhancers
- take UAS, fuse to LacZ gene, and provide a minimal promotor
- introduce into cells that express GAL4 yeast transcription factor
- can detect high level of expression of B-galactosidase

18

lexA/GAL4 domain swap

- activates transcription by binding to the lexA operator in enhancers
- put LexO operator upstream of reporter genes, then add domain swap protein can also detect B-gal but at lower levels than when endogenous GAL4 gene used.
- high enough expression slows domains can function independently and now converted lexA repressor into an activator.

19

GAL4 full domain production of B-gal

lexA - 1800
lexA/GAL4 - 1800

- Same regardless if you also introduce lexA or lexA-GAL4 fusion
- endogenous GAL4 gives high activation of the reporter gene.

20

LexA full domain production of B-gal

lexA - 0
lexA/GAL4 - 0

- good control - with no UAS there will be no activation of B-gal

21

lexA/GAL4 production of B-gal

lexA - 0
lexA/GAL4 - 520

- essentially none because lexA normally a repressor
- 520 because activation domain form GAL4 transcription will be activated.