L5 : Pol II, III and gene regulation Flashcards
What are 5 ways to control gene expression?
- Regulate transcription
- RNA export from nucleus
- Stability (inside/outside nucleus)
- Splicing
- Translational control
How can chromatin modifications allow access to DNA
Condensed chromatin is repressive
- Histone variants
- Chromatin remodellers
- PTMs on tails
How is the general mechanism of transcribing an mRNA gene?
- TF binds DNA (signal integration leading to DNA binding)
- TF interactions with co-activators (histone/DNA mod enzymes, chromatin remodellers)
- TF recruit Pol 2 machinery through co-regulators (mediator complex)
- Pol 2 recruited through the GTF but transcription is regulated
What is the difference between TFs and GTFs?
GTFs - core proteins required for basal transcription initiation by RNAP2
- Assembly at core promoter to form PIC
TFs - Regulatory proteins that control gene expression in a gene-specific manner
- Bind specific DNA seq to activate/repress transcription
What are transcription factors?
Proteins that activate transcription of specific target genes under specific conditions
What is the domain structure and functions of TFs?
- DNA binding domain (DBD)
- Bind sequence specifically to genomic DNA - Signal sensory domains (SSD) - not required
- Regulatory domain that senses external signals (ligand binding or phosphorylation - Activation domains (AD)
- Interact with co-activators to activate transcription - Nuclear localisation signal (NLS)
When do TFs bind?
If present, they may bind:
- As soon as expressed
- After ligand binding
- After phosphorylation
- Release from membrane/inhibitor
Explain nuclear localisation signal?
Monopartite - cluster of 4-8 basic residues (K,R)
Bipartite - two clusters of 2-3 +ve residues separated by 9-12
Signal recognised by nuclear import system (eg. karyopherin)
- Masking or unmasking can allow regulation of TF localisation and activity
Explain DNA binding domain? Examples?
TFs often contain variety of different DBDs that recognise specific DNA sequences
Gal4, Pho4, Gcn4
Where do DNA binding proteins bind?
Usually upstream of ORFs
Can be several binding site
- Far away = enhances
- Closer to TSS = promotes
Experiment to discover binding sites for DBDs?
ChIP-seq (chromatin immunoprecipitation and sequencing)
- Add formaldehyde within cells
- Diffuses across cell and crosslinks
- Lyse cells, nuclease treatment
- Release fragments of DNA cross-linked to protein of interest
What are the features of activation domains?
Difficult to identify by sequence
- Conservation not obvious
- Often highly unstructured and small protein domains with composition bias (eg. acidic-rich, Pro-rich etc)
- Often contain essential bulky hydrophobic residues (W, F, L)
Highly promiscuous in interactions
- Often interact with several co-activators
Become partially structured upon binding coactivator subunit
- Usually forming alpha helix
Explain activation domains?
ADs interact directly with coactivator
Enables activator to stimulate transcription at specific genome location
How can ADs form ‘fuzzy complexes’? Example?
Different ways for activator to interact with co-actviator
eg. transcription activator Gcn4 binds Gal 11 (subunit of mediator complex) in multiple orientations and at multiple sites
Fuzzy complex is dynamic, flexible, disordered
Briefly describe how nrf2 is activated and leads to gene expression
- Needs activation
- Needs NLS
- Needs to bind DNA
- Needs to interact with transcription machinery
Explain the activation and NLS of nrf2?
- As soon as nrf2 is expressed, the protein is degraded by Ub ligase
- ROS inactivates UbL
- Newly synthesised Nrf2 can translocate to the nucleus and activate gene transcription
Explain binding of nrf2 to DNA?
Some repressed promoters are bound by 2 sMafG
- nrf2 binds and replaces 1 sMafG molecule
Some genes repressed by Bach1 and sMafG
- To relieve repression, need hemes that will bind Bach1
- Unmasks nuclear export signal
- Bach1 transported to cytoplasm and degraded by proteasome
- Competition is removed and Nrf2 can then bind
Explain interaction of nrf2 with transcription machinery?
Interacts with mediator complex to recruit Pol 2
Nrf2 can be acetylated, increasing affinity and sequence specifictiy between Nrf2 and DNA
What genes are regulated by Nrf2?
Drives expression of genes involved in:
- oxidative stress response and detoxification (Nrf2 target genes)
- iron metabolism
What are two key observations about Nrf2 domains?
- Multiple binding sites for same TF
- Presence of H3K27 acetylation around Nrf2 binding site
What experiment investigated domains of Nrf2?
ChIP seq
Represents all locations on DNA where Nrf2 can bind
- Can bind at least 3 times in promoter region
- Enhancer region further upstream has other binding sites
- Acetylation of H3 around binding site (Nrf2 recruits CBP/P300)
How do activators and co-activators interact?
- TFs (co-activators) bind genome at specific sites under specific conditions
- Activators subsequently recruit coactivators to genome
- Coactivators can stimulate transcription by multiple mechanisms
Multiple TFs can bind genome and recruit multiple co-activators, allowing very complex programs of transcription
What are 2 examples of co-activators and their function?
Chromatin remodellers
- Remodelling around gene to alter accessibility
- eg. SWI/SNF, ISW1, RSC
Histone modifiers
- Catalytic modification of chromatin (PTMs)
- eg. SAGA, NuA4, CBP/p300
Define epigenetics?
Stably inherited phenotype changes in chromosomes without alterations in DNA sequence
Adds layer of information outside of DNA
