Transcription II

Question 1

Perfect Transcription Has

Answer 1

• nuclear localization signal
• interaction with co-activator
• DNA-binding domain
• transactivation domain (activate RNAPII)
• binding sites for other TFs

Question 2

Test Nuclear Localization

Answer 2

Experiments fused to GFP
- nuclear localization signal (NLS) domain needed
- basic amino acid (+)

Question 3

Test Transactivation Domain

Answer 3

Reporter assay (luciferase + known DBD)
- remove regions of gene until loss of expression

Question 4

Test DNA Binding Domain

Answer 4

Electrophoretic Mobility Shift Assay (EMSA)
- free DNA
- DNA+ protein (higher + thick)
- unlabelled DNA + nonspecific protein (higher + thin)
- mutated DNA + protein (higher + thick)
- antibody + DNA + protein = supershift

Question 5

Co-immunoprecipitation

Answer 5

antibody recognize protein X
maintain interactions –> antibody pulls protein X and other proteins bound to it

Western Blot – targeted (antibody for protein Y)
Mass Spectrometry – general (identify interaction)

Question 6

TF Families = Super Classes

Answer 6

1) Basic
- leucine zipper, helix-loop-helix (MyoD/MYF5 - muscle)

2) Zinc-coordinating DNA-binding domains
- Lim1 = TF in brain development

3) Helix-turn-helix
- homeodomain = specificity (MODY - insulin release)

4) β-scaffold factors with minor groove contacts
- STAT (inflammation), NFAT (immune), P53

5) Orphan TF
- bends DNA facilitate binding other TF