Lecture 4 Flashcards
What is a transcription factor?
Intracellular/nuclear hormone receptors, in which free hormones (steroid, thyroid) diffuse through the plasma membrane of target cells to reach –> regulate transcription
What are the common structural elements in nuclear hormone receptors?
All have a common structure:
- activation domain - stimulate gene transcription
- DNA binding domain - binds DNA of target gene
- ligand binding domain - binds hormone
- other regions responsible for dimerization and translocation
Outline the mechanism of action of steroid hormones
Hormone either:
- binds to receptor in cytoplasm and complex translocates into nucleus
- diffuses into nucleus and binds to its receptors
Regulated by kinases –> phosphorylation of protein
What is the time frame for steroid hormone action?
Slow acting
- takes hours to occur
- much slower than peptide hormones
Describe the mechanism of receptor binding to specific binding sites on hormone responsive genes
AA sequence arranged in a zinc finger formation are located in the region of the H-R complex which bins the hormone responsive genes to stimulate gene transcription
- P box sequence defines specificity of receptor binding to DNA
Briefly describe the gene reporter assay technique
- Promoter of the gene of interest is fused to reporter gene
- gene product is easy to quantify/measure - fluorescent tag - new construct is transfected into cell along with hormone receptor and the cell is stimulated with hormone
- serial deletions or point mutations are made in the promoter to identify which specific parts are responsible for gene activation/repression
- measure activity and delete off regions
- remove activating region –> decrease in activity
- remove another activity region –> loss of activity
- remove repressor region –> a little bit of activity comes back
What is the gene reporter assay used for?
To test the responsiveness of promoter to hormone stimulation in vitro
Describe 3 techniques that can be used to study DNA binding proteins
- DNase foot printing assay
- label double stranded DNA sequence that binds protein factors (promoter) and incubate with binding proteins (nuclear extract)
- incluse control = proteins that don’t bind DNA
- treat with DNase to produce nicks throughout DNA
- separate DNA fragments by size on denaturing PAGE
- sequences that bind proteins will be protected from digestion and bands will not present on gel –> produces hole/”footprint” in h ladder - EMSA
- used to confirm small DNA regions that bind to nuclear proteins
- label DNA fragment suspected of being bound by protein and incubate with cell nuclear extract
- fragments separated by electrophoresis on non-denaturing gel
- heavy DNA protein complexes migrate more slowly than free probe (unbound DNA) = higher molecular weight band - ChIP Analysis
- crosslink DNA binding proteins to DNA
- cut DNA into small fragments
- add antibodies specific to DNA binding protein to isolate complex
- reverse the cross linking to release the DNA and digest the proteins
- use PCR to amplify specific DNA sequences
Discuss the potential role of the nuclear matrix in hormone action
Structural and organizational function
- domains for DNA replication, RNA transcription and splicing
- involved in trafficking of compounds between departments
May direct hormone receptor complex towards specific target gene
- acceptor proteins associated with matrix may bind to a specific domain of the receptor and increase their affinity for responsive elements
Discuss the potential role of the chromatin in hormone action
Chromatin = string of nucleosome, DNA wrapped around a core of histones (active = highly acetylated)
- histone methylation –> either activate or repress gene transcription
- methylated AA recognized by enzymes that influence transcription
Main factor responsible for allowing transcription of certain genes is the compactness of DNA –> chromatin
- when hormone receptor complex binds to DNA - recruits and interacts with TFs –> activates RNA polymerase
Describe experiments to identify DNA sequences involved in hormonal regulation of gene expression
- gene reporter assay
- DNase foot printing assay
- EMSA
Describe a mechanism of how different hormone responses can be integrated
Steroid hormones can also have non-genomic effects and integrate with the effects of peptide hormones:
- steroid hormones can affect the activity of protein hormones by increasing synthesis of receptors, protein kinases or phosphoprotein phosphatases
