W12L2 Flashcards
Transcription factors
Over 2,000 transcription factors identified to date
General Transcription Factors
- initiates gene expression
- TFII proteins
- Bind all genes
- RNA polymerase II is NOT considered a transcription factor
Tissue Specific Transcription Factors
- increases transcription rate and efficiency for a specific gene
- binds to a subset of genes in a cell and time specific fashion
- is required for complete expression of a gene
What does a perfect transcription factor have?
Not all transcription factors have these:
Nuclear localization signal
DNA binding domain
Bindings sites for other TFs
Interaction with co-activator
Transactivation domain (TAD)
Nuclear localization
Nuclear Localization Signal (NLS)
- a short domain of the protein is required for nuclear localization
Identified with Green Fluorescent Protein
Different Types of Nuclear Localization Signals
- they are generally basic or positive charge
- DNA is negative
- All have basic amino acids – lysine (K), Arginine (R)
Transactivation domain (TAD)
TAD works by itself or targeted to a different protein
Identified with Luciferase
Reporter assay to assess Transcription Assay Domain
- Need a reporter gene that contains the site that the protein will bind to (DNA binding domain)
DNA binding domain
Identified with Electrophoretic Mobility Shift Assay (EMSA)
Radiolabel a known sequence of DNA (20-30 bp)
Mix with purified protein
Interaction leads to a “shift”
Shift is lost when adding excess unlabeled sequence
- putting un-radiolabelled DNA probe is a type of control
Supershift when you include an antibody against the protein
Co-activator or other TFs
Identified with co-immunoprecipitation to show binding of co-activator or other TFs
Suggest that two proteins interact
Purify one proteins from an extract with an antibody specific to that protein
MAINTAIN INTERACTING proteins
Assess what other proteins are pulled out by the first protein
Assess by western blot or mass spectrometry
Transcription factor families
Often classified based on their protein binding motifs (binds from smaller to larger regions of DNA; aka binds from more specific roles to more general roles)
Super classes:
- Basic
- Zinc-coordinating DNA-binding domains
Transcription factor families
Often classified based on their protein binding motifs (binds from smaller to larger regions of DNA; aka binds from more specific roles to more general roles)
Super classes:
- Basic
- Zinc-coordinating DNA-binding domains
- Helix-turn-helix
- Beta-scaffold factors with minor groove contacts
- Orphan transcription factors
Basic transcription family
- Leucine zipper (ZIP)
- Helix-loop-helix (HLH)
- bHLH proteins bind the consensus
DNA sequence CANNTG
- MyoD and MYF5 are bHLH proteins expressed in myogenic precursors
— Loss of both results in no muscle - HLH-ZIP
Zinc-coordinating DNA-binding domains transcription family
- Zinc fingers or zinc clusters
- Provide specificity for DNA sequences
LIM proteins composed of two contiguous zinc fingerdomains
- Name based on motif found in Lin-1, Isl-1 and Mec-3
- Lim1: Transcription factor expressed during brain development
Helix-turn-helix transcription family
Consists of:
- Homeodomain (specificity of DNA recognition lies within the homeodomain)
- Paired Box
- Fork Head
Regulates the insulin gene
MODY (Maturity onset diabetes of the young)
- 5-10% of those patients with type II Diabetes get it young
- 6 different MODY types, and mutating it will get insufficient insulin
- autosomal dominant
- these MODYs were identified as genetic determinants of diabetes before the specific genes were known
Beta-scaffold factors with minor groove contacts transcription family
- Includes the TFII transcription factors
Are transcription factors that bind a lot of genes to open up chromatin and stabilize it
STAT (signal transducer and activator of transcription)
Involved in inflammation and cancer
Trigger by jun kinase (JNK)
NFAT (Nuclear factor of activated T-cells)
Involved in immune response
Triggered by increased calcium
P53
Represses the cell cycle
Mutated in many cancers
Involved in DNA damage repair
Orphan transcription factors transcription family
HMGB
Bends DNA to facilitate binding of other
transcription factors (such as p53)
