What does a DNA control element do?
Acts locally (cis)
Binding of trxn factors to a DNA control element regulates the gene the control element is associated with.
What are two broad classes of gene expression regulation?
- Control Elements - act locally - cis
2. Trxn’l Activators and Repressors - act at a distance - trans
Give three examples of DNA control elements.
- TATA box
- Promoter Proximal Elements
Where is the TATA box (initiator seq.) and what does it do?
23-35 bp upstream of trxn start site 20 bp long determines site of trxn initiation directs/binds RNA pol II binds general trxn factors (TF II ?)
What are basal trxn factors?
Base trxn factors that bind to all promoters to recruit in the rest of the trxn machinery & RNA pol II
What are Promoter Proximal Elements?
located 200 bp upstream of trxn start site
8-20 bp long
Help to regulate trxn
are bound by cell specific factors (or by factors found in all cell types)
What would happen if you moved a promoter proximal element kilobases upstream (or downstream)?
Would not function properly.
PPE’s need to be relatively close to the gene they are regulating (200 bp)
What are enhancer elements?
can be 200 - 10kb up or down stream of start site OR within an intron
is 100-200 bp long but made up of many small (8-20bp) control elements (binding sites for different factors).
are often cell-type specific
Give three examples of diseases arising from mutations in DNA control elements.
- Beta-Thalassemia or (gamma-delta-beta) Thalassemia
- Hemophilia B Leyden
- Fragile X-Syndrome
Give two examples of Transcriptional Activators / Repressors
- Sequence specific DNA binding proteins
2. Co-factors - bind to the DNA binding proteins (not to the DNA itself)
How do sequence specific DNA binding proteins do their job?
They bind to a specific sequence (DNA control element !) 6-8bp longInsert alpha-helix into MAJOR groove of DNAContact between aa side chains and DNA bases
Distinguish between major and minor groove binding.Give an example of a trxn protein that binds the minor groove
Major groove binding is very sequence specific. Minor groove binding will distort the DNA more.The TBP (TATA binding protein) binds to the minor groove using the beta sheet.
TATA box and bends it to a right angle, inserts aa’s in between base pairs, thus partially unwinding DNA. So it has a small amount of melting activity.
What is modularity in sequence-specific DNA binding proteins?
have specific domains that do specific things. if you swap out a module from one gene into another gene, the second gene will gain the activity of the inserted module
Name 3 specific homeodomain proteins (genes)
- Hox family - regulates anatomical body plan (antennapedia mutation)
- Pit1 - pituitary specific - growth hormone regulation
- Msx - neural crest cells responsible for proper craniofacial development
What is the general geometry of a homeodomain sequence specific DNA binding protein? Where does it bind?
Helix - Turn - Helix
Binds to the major groovetwo alpha helices connected by a turn of DNA, running antiparallel to one another
A third alpha helix connected by a turn of DNA, running perpendicular to the first two
Give some examples of zinc-finger sequence specific DNA binding proteins.
- Estrogen receptors
- Androgen receptors
- retinoic acid recptor
What is the general geometry of a zinc finger DNA binding domain?Where does it bind (usually)?
Zinc ion coordinate by either 4Cys residues or 2Cys2His residues
Fingerlike projections bind to DNA
—->recall His is the imidazole (Nitrogen containing) amino acid
—> recall Cys is -CH2-SH
Give two examples of the bZIP (basic zipper) sequence-specific DNA binding proteins.
- c-fos - heterodimer. over expressed in some cancers
2. c-jun - hetero or homodimer - biologically significant in some cancers
Describe the general geometry of the bZIP family of sequence specific DNA binding proteins.
Basic amino acid roughly every 7 aa’s (ex. leucine).
Dimerizes (homo or hetero)
Give 3 examples of basic helix-loop-helix (bHLH) sequence specific DNA binding proteins
- MyoD - muscle differentiation
- myogenin - skeletal muscle development (myogenesis & repair)
- Myf5 - muscle differentiation (myogenesis)
Describe the general geometry of the bHLH class of sequence specific DNA binding proteins.
A dimer with two helices each with a small loop interrupting the helix in the middle(ish).
Contains a basic amino acid that binds the DNA.
Give 4 classes of sequence specific DNA binding domains
- Homeodomain (HTH)
- Zinc Finger
What are 3 diseases caused by mutations in seq. specific DNA binding domains and which domain is each associated with?
- Craniosyostosis - MSX 2 gene. Homeodomain. Sutures close prematurely
- Androgen Insensitivity Syndrome (AIS) - androgen receptor (dna binding domain or ligand binding domain) - Zinc Finger. XY genotype, female external phenotype.
- Waardenberg type II - MITF gene - bHLH - melanocytes distupted
Why is dimerization important?
Allows for more variability in DNA binding specificity of binding domains.
Once bound to DNA, what do Activators and Repressors do?
Regulate assembly of initiation complexes and rate of trxn initiation
What else can Activators and Repressors do (besides reg. initiation complex)?
Affect chromatin structure influencing the ability of trxn factors to bind promoters.
Which DNA seq. specific binding proteins can form heterodimers and how is this helpful?
bZIP, bHLH, zinc finger (why not HTH?)
“Combinatorial Control” - more options for binding
Why is HTH not a heterodimer?
It is one continuous strand forming the helix - turn - helix super-tertiary structure.
So, not a dimer b/c not two different things coming together.
How do activators / repressors get the job done once bound to DNA?
regulate recruitment trxn initiation complexes
regulate rate of trxn