Domain Swaps in Molecular Genetics

Question 1

Domains

Answer 1

• dimerization
• activation
• DNA binding

Question 2

Domains are Distinct

Answer 2

• 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

Question 3

LexA gene

Answer 3

• repression

Question 4

GAL4 gene

Answer 4

• activation

Question 5

lexA/GAL4

Answer 5

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

Question 6

Recombinant protein

Answer 6

They are derived by procedures involving recombinant DNA.

Question 7

Enhancers

Answer 7

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

Question 8

regulatory region of genes

Answer 8

• where transcription factors interact with genes

Question 9

upstream activating sequence

Answer 9

• DNA binding domain interacts with

Question 10

transcription factor

Answer 10

• diffusible protein made elsewhere.

- most transcription factors bind as heterodimers

Question 11

dimerization domain

Answer 11

• will bring two GAL4 together (dimerize)

Question 12

activation and dimerization domain

Answer 12

• amino acids in these are covalently linked

Question 13

DBD and DNA

Answer 13

• interactions between these two are non-covalent.

Question 14

Silencers

Answer 14

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

Question 15

Domain swap

Answer 15

• switch DBD of lexA with GAL4

Question 16

Test using reporter genes

Answer 16

• 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

Question 17

GAL4

Answer 17

• 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

Question 18

lexA/GAL4 domain swap

Answer 18

• 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.

Question 19

GAL4 full domain production of B-gal

Answer 19

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.

Question 20

LexA full domain production of B-gal

Answer 20

lexA - 0
lexA/GAL4 - 0

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

Question 21

lexA/GAL4 production of B-gal

Answer 21

lexA - 0
lexA/GAL4 - 520

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