Eukaryotic transcriptional regulation (lecture 4) Flashcards
human protein coding genes vs non coding?
20 000 - 25 000 protein-coding genes
this is only 1.5% of the genome!
(pro: only coding sequences)
Steps at which eukaryotic gene expression can be controlled
transcription
RNA processing
RNA transport from nucleus to cytosol
translation
enhancer/silcencer features
- Regulatory DNA sequences (ca. 4-20bp)
- Located even thousands bp from the start of transcription
- Located upstream, downstream, or even within the gene they
control. - Function also in opposite orientation
- Binding sites for transcription factors (activating/repressing)!!
how can one study enhacers/silencers?
- put the corresponding promotor + at least -2kb in front of a reporter gene
- reporter: luciferase enzyme, gfp
- perform promoter deletion assay/promoter ponit mutation assay
- if important sequences are affected one will observe an dramatic decrease in expression of the reporter gene
how to you ensure that it is an enhancer/silencer?
clone it beyond its original context and proof it again
enhancers work independetly of its promoter context
types of TFs
1) True Activators/Repressors
a) directly b)indirectly
- Contain DNA binding domain and activation/repression domain
- Establish contact with basal transcription machinery
- Activity on DNA or chromatin templates
2) Antirepressors/activators
- Recruit chromatin remodeling complex or histone modifiers
- > only on chromatin templates and NOT DNA templates
3) Architectural proteins (HMG (high mobility group))
- Determine DNA topology (bending)
- in that way may facilitate or prevent protein complex formation
Architectural proteins: types and examples?
Canonical HMG proteins HMG‐1 and HMG‐2:
§ Multiple HMG domains
§ Do not bind to any specific DNA sequences.
§ Non-canonical HMG proteins as SRY, SOX, TCF‐1 and LEF‐1:
§ Single HMG domains
§ Bind to specific DNA sequence motifs.
§ Specific, important and well‐defined biological roles, particularly in embryonic
development.
e.g. IFN beta promoter
types of DNA binding domains in TFs
Helix–turn-helix
Zinc fingers
Helix–loop-helix
Leucine zippers
Heterodimerization can alter DNA-binding specificity
method EMSA
Electrophoretic mobility shift assay
analysis of DNA-Protein interactions (shift = interaction)
supreshifts can be achieved by antibodies
Chip assay
chromatin immuno precipitation assay
analysis of DNA-protein interactions
you can pull down proteins with anti-tag-antibodies on beads (by centrifugation or magnetic forces) and see if distinct DNA fragments come with it
-> afterwards you can do protein and DNA analysis
GAL4-UAS assay system
GAL4 is an activating TF:
binding domain: GAL4 BD
activation domain: GAL4 AC
UAS is the corresponding promoter
you clone this system in front of an reporter gene like luciferase
you can check if protein X has an activator/repressor domain for transcription machinery ->
GAL4-UAS Transactivation-Based Assay
you can check if protein X has a binding domain for a certain sequence/promoter
-> GAL4 DNA binding-based assay (X is linked to AD)
Mechanism of action of true activators or repressors
how do they achieve their spatial or temporal specificity?
a. Tissue specific expression/location (e.g. homeodomain proteins)
b. Covalent modification (e.g. HSF (heat shock TF))
c. Ligand binding (e.g. steroid receptors)
d. Sequestering in the cytoplasm (e.g. NFKB)
e. Sequestering by repressor (e.g. HLH proteins)
f. Activation by cleavage of inactive form
what is chromatin?
Histones
Nonhistones (HMG, TFs)
DNA
maximum package in a chromosome?
10 000 fold shorter than extended
what are nucleosomes?
beads-on-a-string form of chromatine
histone octamer and 147 bp long DNA strand
dynamic structure
