Flashcards in Domain Swaps in Molecular Genetics Deck (21):
- DNA binding
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
- fusion protein
- DBD - lexA
- activation/dimerization - GAL4
They are derived by procedures involving recombinant DNA.
- contain multiple binding sites for transcription factors
- binds activator
- GAL4 doesn't work well unless hooked up to an enhancer and enhancer-binding protein
regulatory region of genes
- where transcription factors interact with genes
upstream activating sequence
- DNA binding domain interacts with
- diffusible protein made elsewhere.
- most transcription factors bind as heterodimers
- will bring two GAL4 together (dimerize)
activation and dimerization domain
- amino acids in these are covalently linked
DBD and DNA
- interactions between these two are non-covalent.
- bound by repressors
- bind to lexO
- DNA sequences bound by lexA differ from sequences within upstream activating sequence bound by GAL4
- switch DBD of lexA with GAL4
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
- 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
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.
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.
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