MBB 267 Week 9: Mitchel 6 Flashcards
What are the key features that express genes?
The effective concentration, availability and activity of sTFs are key to the expression of a given gene.
How is the activity of transcription factors regulated?
the activity of a transcription factor can be regulated in a number of different ways:
(i) de novo synthesis;
(ii) binding of a ligand molecule;
(iii) post-translational modification, e.g. phosphorylation; (iv) formation of a protein complex;
(v) release from an inhibitor molecule;
(vi) proteolytic activation.
When does the cell regulate transcription?
often regulated in response to extracellular stimuli that control growth, differentiation, homeostasis and communication
Generally, how do signals affect transcription?
Signalling molecules can bind to transmembrane receptors, activating a signal transduction pathway, or pass through the plasma membrane and bind directly to the transcription factor
What is a nuclear receptor?
When the receptor is itself the specific transcription factor
What is the common structure of nuclear receptors?
Nuclear receptors have a common structural organisation, comprising a highly conserved central C4 zinc finger DNA-binding domain (see lecture 3), a structurally conserved C-terminal ligand-binding domain, and an N-terminal activation or repression domain of variable structure. Nuclear receptors have C4 zinc finger domains and function as homomeric or heteromeric dimers
How do nuclear elements work?
Nuclear Receptors contain 2 C4 zinc finger domains (“P box” and “D box”). (Proximal box for DNA/protein interactions and Distal box is for protein/protein interactions)The P box binds the response element (RE); the D box mediates dimerisation.
- Homodimeric NRs (e.g. estrogen receptor, glucocorticoid receptor) bind palindromic repeats.
- Heteromeric NRs (e.g. retinoic acid receptor) bind direct or inverted repeat sequences and contain a common monomer, the retinoid X receptor (RXR).
How are homodimeric nuclear receptors activated?
Homodimeric receptors are localised in the cytoplasm in the absence of their ligand. They are anchored in complexes with heat shock proteins, meaning they are stuck and cannot enter the nucleus. Upon receptor entry into the cell, hormone binds to its receptor. Hormone binding releases the receptor from its inhibitor and allows dimerisation. The ligand/receptor complex then enters the nucleus
How are heterodimeric nuclear receptors activated?
Heterodimeric receptors are localised only in the nucleus, and contain the retinoid X receptor (RXR) and occupy the response element. In the absence of a ligand, bound corepressors recruit HDACs which block transcription. Ligand binding displaces corepressor protein and allows recruitment of HAT complexes and interacts with mediator complex which allows transcription to take place.
How do signalling pathways transduce signals to switch on transcription?
3 ways;
- The receptor can directly activate the transcription factor
- transduction of the signal can involve multiple steps
- a secondary signal molecule can be generated within the cytosol
How do signalling pathways work?
Signalling pathways normally involve multiple steps and generate a biochemical cascade. The pathways are linked through cross- talk to generate signalling networks that allow coordinated responses and combinatorial regulation. Different cell types respond to a given signal in different ways. An associated protein kinase is activated, resulting in activation of a transcription factor. Signalling pathways are well conserved. There is extensive crosstalk, allowing coordinated responses and combinatorial effect
What is the JAK/STAT pathway?
JAKs are protein kinases associated with cytokine receptors. Cytokines (eg interleukins, interferon, erythropoietin) are important for cell growth and differentiation. Cytokine binding to its receptor induces dimerisation. As a result, the JAK kinases are brought close enough together to phosphorylate one another at a critical tyrosine residue in a structural feature of the protein known as the activation lip. Phosphorylation reduces the dissociation constant for ATP or the substrate (higher affinity), leading to increased kinase activity. The activated JAK kinases then phosphorylate several residues on the receptor. Receptor phosphorylation leads to binding of STAT (signal transduction and activation of transcription) transcription factors through their SH2 domains, that are then phosphorylated by the JAK kinase. The phosphorylated STAT dissociates from the receptor and dimerises. Dimerisation exposes a nuclear localisation signal (NLS), enabling the protein to be imported into the nucleus
How do receptor tyrosine kinases signalling pathways work?
Receptor tyrosine kinases (RTKs) have intrinsic kinase activity and are activated by protein hormones (e.g. insulin) and growth factors (e.g. fibroblast growth factor, epidermal growth factor). Receptor dimerisation triggers autophosphorylation. Human cells express 4 distinct RTKs for epidermal growth factor named HER1, HER2, HER3 and HER4. Ligand binding causes a surface loop to be extended, providing the dimerisation interface. The HER2 receptor does not bind ligand but exists as a monomer in the preactivated conformation primed for dimerization and acts to potentiate the activity of the other HER receptors. Amplification of the HER2 gene occurs in 25 % of breast cancer patients, resulting in ~ 100-fold increase in the level of HER2 receptor. The drug herceptin binds the extended loop conformation, blocking formation of heterodimers
How does the Ras/MAP kinase pathway work?
Almost all RTKs activate the Ras/MAP kinase pathway. The Ras protein is a small GTPase molecular switch protein. These proteins are in the active state when bound to GTP and inactive when bound to GDP. They are “switched on” by guanine nucleotide exchange factors (GEFs) and “switched off” by GTPase activating proteins (GAPs). Ras triggers a kinase cascade, ending in activated MAP kinases that enter the nucleus and target sTFs. Ras, Raf and many RTKs were identified as oncogenes
How is CREB activated?
The transcription factor CREB (cyclic AMP response element binding protein) is activated through the cAMP/Protein kinase A pathway, which is induced by G-protein coupled receptors (GPCRs). GPCRs have seven transmembrane receptors and are associated with a cytosolic heterotrimeric GTPase. Binding of the ligand enables the receptor to function as a GEF for the GTPase. In the GTP-bound (active) form, the a subunit is released and can activate the membrane-bond adenylyl cyclase enzyme. Adenylyl cyclase converts ATP to cAMP. Increased cytosolic cAMP levels cause the release of the catalytic subunit of protein kinase A (PKA) from its repressor subunit. The catalytic subunit of PKA can then translocate into the nucleus and activate CREB through phosphorylation. Phosphorylation of CREB stimulates its interaction with its coactivators.
