EXAM I White Flashcards
How does the leucine zipper motif bind to DNA?
Dimerizes through leucine zipper region
Through interactions b/w hydrophobic aa side chains = leucines
What are the 4 DNA-binding domain structural motifs?
Helix-turn-helix
Zinc finger
Leucine zipper
Helix-loop-helix
Which DNA-binding motifs fits into the major groove of DNA?
Helix-turn-helix
Zinc-finger
Describe the helix-loop-helix domain
short alpha chain connected by a loop to a second longer alpha chain
Homodimers or heterodimers
Explain the use of EMSA in the identification of TFs; What is it used for?
What are other ways in which you can identify TFs?
Used to detect sequence-specific DNA binding proteins
Gel mobility shift assay
Electrophoretic mobility shift assay
- Affinity Chromatography
- Isoloate DNA binding protein
- Purification
- CHIP: Chromatin ImmunoPrecipitation
What is CHIP used for in identifying TFs?
To identify sites in the genome that a known regulatory protein bind to
T/F Histone acetylation makes it easier to remove histones
TRUE
Opposite for methylation = heterochromatin
In what ways do gene repressors inhibit transcription?
- Competitive DNA binding (both activator and repressor bind)
- Masking of activation site (repressor binds to activation domain)
- Direct interaction with the general TFs (repressor binds to DNA and blocks assembly of general TFs)
- Recruitment of chromatin remodeling complexes (repressor recruits the complex returning the promoter to pre-transcriptional nucleosome state)
- Recruitment of histone deacetylases (harder to remove deacetylated histones and open up DNA)
- Recruitment of histone methyl transferase - (chromatin is silent)
How are iron responsive elements (IREs) and iron responsive regulatory proteins (IRP) involved in mRNA?
During iron starvation - IRP binds IRE at 3’ = transferrin receptor made
During excess iron - IRP binds IRE 5’ = ferritin made
How does proteosome recognize proteins required for destruction? Describe the mechanism
Ubiquitin
Ubiquitin binds to Cysteine side chain E1 enzyme
Transferred to E2 ubiquitin ligase (complex then marks proteins for destruction)
Protein then adds to ubiquitin ligase and ubiquitin chain binds to lysine on the protein
Continuous addition of ubiquitin chain on lysine = recognized by proteasome
DNA methylation causes ______ of gene expression
Repression
Used by genomic imprinting
XIC - X-inactivation center
What are the 7 processes in which gene regulatory proteins are controlled?
- Synthesis
- Ligand Binding
- Covalent modification-phosphorylation
- Addition of subunit
- Unmasking
- Stimulation of nuclear entry
- Proteolysis
Explain the role of 5’ cap and poly A tail in mRNA expression
- Poly-A Tail - confers stability. Poly-A tail is shortened by exonuclease, which acts as a timer. Once its reduced to 25nt, two pathways converge to degrade mRNA (i.e. half life; globin)
- 5’cap - protects the RNA from RNA degrading enzymes. If decapping occurs, the exposed mRNA is degraded from the 5’end
Give examples of post-translational modifications
Modified by protein kinases
- Covalent modifications (i.e. acetylation, phosphorylation)
- Non-covalent modification (folding)
- The binding of other protein subunits
Explain the experiment that lead to the conclusion that TFs are modulators
By using a protein that contains the activation domain and DNA binding domain. Mutant proteins that contained various domains were deleted and with each progressive deletion, you had a loss of functionality. This experiment showed that TFs contain domains that are functional
