BIMM 100: LE's 13 – 15 questions well
answered graded A+
expression of a gene ultimately depends on what 2 things? - correct answer ✔✔ - the nuclear
concentration/availability and activity of a specific set of TFs bound to the gene regulatory
regions (proximal promoter elements and enhancers/silencers)
what is used to evaluate TF promoter occupancy (protein-DNA interaction) 'in vitro'? - correct
answer ✔✔ - EMSA and DNase foot printing
what is used to evaluate TF promoter occupancy (protein-DNA interaction) 'in vivo'? - correct
answer ✔✔ - chromatin immuno-precipitation (ChIP) assays
ChIP assay - steps (5) - correct answer ✔✔ 1) fix the chromatin in living cells with formaldehyde
to keep the proteins and DNA stuck together
2) fragment via sonication
3) purify and immunoprecipitate the chromatin with an antibody to recognize and isolate the TF
of interest
4) reversal of cross links (unsticking the proteins and DNA from one another)
5) analysis of the immunopurified DNA sequences
ChIP assay: glucocorticoid receptor example (purpose, steps (2), results) - correct answer ✔✔ -
monitor the binding of the glucocorticoid to the IkB-alpha promoter
1) ChIP for the receptor
2) do PCR for the IkB-alpha promoter
- when there are no glucocorticoids present, no PCR product is expected because if there is no
GR bound, there is no transcription
, - when there are glucocorticoids present, PCR product is expected because if GR is bound,
transcription will occur
what are the 2 molecular mechanisms for transcriptional activation or repression? - correct
answer ✔✔ 1) recruitment of RNA pol II to the core promoter region
2) chromatin modification and remodeling
recruitment of pol II to core promoter region - correct answer ✔✔ - activators interact with the
mediator complex to promoter the assembly of the transcription pre-initiation complex at the
core promoter region
mediator complex (what it is and the main idea) - correct answer ✔✔ - a multi-subunit co-
activator complex that interacts with RNA pol II
- main idea: works as a molecular bridge between transcription activators and the RNA pol II
activation domains - correct answer ✔✔ domain on the activator TFs, where they'll bind to the
mediator and begin transcription
transcription initiation is a __________ ____________ process. why? - correct answer ✔✔ -
highly cooperative
- b/c many RFs promote and are a a part of the pre-initiation complex
transcription pre-initiation complex assembly (3) - correct answer ✔✔ 1) general TFs and
activators bind to the core promoter, proximal promoter, and enhancer elements
2) the coactivator mediator complex is recruited by activation domains and TAFs
3) RNA pol II is recruited by interactions with the mediator complex and TFII factors
chromatin modifications (2) - correct answer ✔✔ 1) histone modification
2) nucleosome remodeling
answered graded A+
expression of a gene ultimately depends on what 2 things? - correct answer ✔✔ - the nuclear
concentration/availability and activity of a specific set of TFs bound to the gene regulatory
regions (proximal promoter elements and enhancers/silencers)
what is used to evaluate TF promoter occupancy (protein-DNA interaction) 'in vitro'? - correct
answer ✔✔ - EMSA and DNase foot printing
what is used to evaluate TF promoter occupancy (protein-DNA interaction) 'in vivo'? - correct
answer ✔✔ - chromatin immuno-precipitation (ChIP) assays
ChIP assay - steps (5) - correct answer ✔✔ 1) fix the chromatin in living cells with formaldehyde
to keep the proteins and DNA stuck together
2) fragment via sonication
3) purify and immunoprecipitate the chromatin with an antibody to recognize and isolate the TF
of interest
4) reversal of cross links (unsticking the proteins and DNA from one another)
5) analysis of the immunopurified DNA sequences
ChIP assay: glucocorticoid receptor example (purpose, steps (2), results) - correct answer ✔✔ -
monitor the binding of the glucocorticoid to the IkB-alpha promoter
1) ChIP for the receptor
2) do PCR for the IkB-alpha promoter
- when there are no glucocorticoids present, no PCR product is expected because if there is no
GR bound, there is no transcription
, - when there are glucocorticoids present, PCR product is expected because if GR is bound,
transcription will occur
what are the 2 molecular mechanisms for transcriptional activation or repression? - correct
answer ✔✔ 1) recruitment of RNA pol II to the core promoter region
2) chromatin modification and remodeling
recruitment of pol II to core promoter region - correct answer ✔✔ - activators interact with the
mediator complex to promoter the assembly of the transcription pre-initiation complex at the
core promoter region
mediator complex (what it is and the main idea) - correct answer ✔✔ - a multi-subunit co-
activator complex that interacts with RNA pol II
- main idea: works as a molecular bridge between transcription activators and the RNA pol II
activation domains - correct answer ✔✔ domain on the activator TFs, where they'll bind to the
mediator and begin transcription
transcription initiation is a __________ ____________ process. why? - correct answer ✔✔ -
highly cooperative
- b/c many RFs promote and are a a part of the pre-initiation complex
transcription pre-initiation complex assembly (3) - correct answer ✔✔ 1) general TFs and
activators bind to the core promoter, proximal promoter, and enhancer elements
2) the coactivator mediator complex is recruited by activation domains and TAFs
3) RNA pol II is recruited by interactions with the mediator complex and TFII factors
chromatin modifications (2) - correct answer ✔✔ 1) histone modification
2) nucleosome remodeling
