What are the different eukaryotic DNA control elements?
TATA Boxes (initiator sequences), promoter proximal element, enhancers
What is a TATA Box (initiator sequence)?
The site at which transcription factors bind, 25-35 base pairs upstream from the transcription start site. It determines the site of transcription initiation and directs the binding of RNA pol II.
What is a promoter proximal element?
A 20 base pair sequence that is approximately 200 base pairs upstream from the transcription start site. Depending on the cell type, specific factors might bind to it.
What is an enhancer?
A series of nucleotides, upstream or downstream, consisting of multiple control elements totaling 100-200 bps. Each control element is 8-20 bps in length. Can be very far away.
What are three diseases that arise from mutations in DNA control elements?
ThalassemiasHemophilia B LeydenFragile X Syndrome
Describe the mutation that leads to thalassemia and its clinical presentation.
A mutation in the promoter of the ß-globulin gene. This results in less ß-globulin protein. A patient would present with clinically mild” symptoms including tiredness, fatigue, and shortness of breath
Describe the mutation that leads to Hemophilia B Leyden and its clinical presentation.
A mutation in the promoter of the Factor IX gene. Less Factor IX is produced (1% when young). This negatively affects the ability of the body to form blood clots. X-linked and effects young males most.
Describe the mutation that leads to Fragile X Syndrome and its clinical presentation.
Caused by excess CG repeats in the 5’ region of the FMR1 gene which leads to transcriptional silence of gene. Causes mental retardation, dysmorphic facial figures, post pubertal macro orchidism.
What roles do transcriptional activators and repressors perform?
Activation and repression of transcription!But you should know that these are proteins made from other genes.
What are the two classes of activators and repressors?
Sequence specific DNA binding proteinsCo-factors
How do sequence specific DNA binding proteins work?
They bind to promoter enhancer elements that are 6-8 base pairs long on the target genes. They bind DNA by inserting alpha helices into the major groove. AA R groups contact base pairs of DNA to regulate transcription.
How do co-factors work?
They bind to sequence specific DNA binding proteins (not DNA) and affect transcription through this contact.
What are the two domains of a sequence specific DNA binding protein?
DNA binding domain: confers sequence specificityActivation domain: mediates protein-protein interactions and recruits general transcription machinery/co-factors
What are the four major families of sequence specific DNA binding proteins?
Homeodomain (helix-turn-helix),Zinc Finger,Basic Leucine Zipper (bzip),Helix-Loop-Helix
How does one go about categorizing sequence specific DNA binding proteins?
Based on the structures of their DNA binding domain!
What are three diseases that can arise from a mutation in a sequence specific DNA binding protein?
CraniosynostosisAndrogen Insensitivity SyndromeWaardenburg Syndrome Type II
Describe the mutation that leads to craniosynostosis and its clinical manifestations.
A mutation in homeodomain MSX2 causes it to bind too strongly to DNA. This affects the transcription of other genes critical for suture closure leading to craniosynostosis (pre mature fusion of forehead).
Describe the mutation leading to androgen insensitivity syndrome and its clinical manifestations.
Mutations in the either the DNA binding domain or activation domain in the androgen receptor protein leads to feminization and under masculinization.
Describe the mutation that leads to Waardenburg Syndrome Type II and its clinical manifestations.
Mutation in the MITF gene that encodes for a transcription factor that plays a major role in the development of melanocytes leads to deafness and pigmentation anomalies of hair and skin.
What is combinatorial control?
Thousand of transcription factors can come together in different ways to regulate thousands of genes differentially.
List the two classes of chromatin remodeling factors and describe how they work.
DNA dependent ATPases that disrupt histone octomers and DNA.Factors that reversibly modify histones through acetylation via histone actetyltransferases (HAT) + histone deacetylases (HDAC)
How does HAT activity influence translation?
Acetylation of lysine allows for the binding of specific transcription factors and once histone is acetylated it recruits transcription factors. HATS are co-activators.
How do HDACs influence transcription?
In the presence of HDACs histones retain positive charge and this maintains its interaction with DNA which prevents access of transcription factors to promoters. HDACs are co-repressors.
What are two diseases in which histone acetylation is altered?
What mutation leads to Rubinstein-Taybi Syndrome and what are its clinical manifestations?
A mutation in the Creb Binding Protein, a HAT (co-activator), leads to widespread transcriptional changes during development.Growth retardation + mental retardation + craniofacial dysmorphism.
How do activators/repressors modulate transcription…On polymerase II + transcription factors?Chromatin?
Interact with pol II + transcription factors to initiate/block elongation of primary transcript.Interact with chromatin to regulate accessibility of DNA to pol II.
What are the basic principles of transcriptional regulation (4)?
- Specificity of activators/repressors to DNA control elements.2. DNA-protein, protein-protein regulating interactions.3. Interactions affecting conformation of DNA (HAT/HDAC).4. Control is combinatorial.
What are the 5 mechanisms in which DNA binding proteins are regulated?
- The conformation of the DNA-binding protein can be altered by ligand binding2. Entry into the nucleus can be regulated3. The amount of transcription factor in the cell can be regulated4. DNA binding can be regulated5. Phosphorylation of the DNA-binding protein can alter various properties
How is the activity of nuclear hormone receptors regulated?How does tamoxifen work?
Estrogen binds to nuclear hormone receptors and activates proliferative cell types. This can be dangerous in cancer. Tamoxifen antagonizes estrogen by binding to estrogen receptors and preventing recruitment of HAT cofactors. Or, it can recruit co-repressors in some cell types.
What is an example of a sequence specific DNA binding protein (SSDBP) being regulated by nuclear entry?What is the mechanism?
In the stomach, Nf-Kb is a SSDBP that enters the nucleus to turn on an inflammatory response when needed.Normally it is sequestered in another protein, IkB which gets ubiquinated and degraded to free nf-kb. Nonpathogenic salmonella can interfere with the ubiquitination of Ikb and thus blocks an inflammatory response.
How can the amount of an activator/repressor be regulated in a cell?
Consider Beta catenin (BC). In the absence of WNT signaling, it is to be degraded in a ubiquitin-proteosome pathway. In the presence of WNT signaling, it is not degraded, its [cytoplasmic] rises, and it goes to the nucleus and triggers expression of WNT responsive genes.
How can DNA binding of a SSDBP be inhibited?
An example - Id proteins: The Id family members negatively regulate DNA binding by heterodimerizing with other HLH proteins through their HLH domains, but preventing DNA binding due to their lack of a basic domain. Relative abundance of these proteins is what dictates transcription or not.
What is an example of a protein modification that can alter the activity of a SSDBP?What is the mechanism?
When the CREB protein is interacting with DNA, it needs to be phosphorylated to promote transcription. It becomes phosphorylated when a ligand binds its guanine nucleotide binding protein. Once phosphorylated, it recruits a HAT, Creb Binding protein, and RNA pol II.
What are four other mechanisms (besides transcriptional regulation) to control levels of gene expression?
- Control of mRNA export from the nucleus2. Control of mRNA degradation3. Control of efficiency of translation4. Control of protein degradation