Lecture 8: Control of Gene Expression in Eukaryotes: Part I Flashcards
- DNA regulatory elements - Regulatory proteins, domain organisation and DNA -binding structures
1
Q
Describe when eukaryotic gene expression is controlled
A
level of initiation of transcription
- by affecting either:
- the formation of the PIC
- regulating local chromatin structure
2
Q
Describe how regulatory regions where first identified
A
- Regulatory elements in promoters were identified -> systematic replacement of short DNA segments with a DNA linker containing a random sequence of exactly the same size
3
Q
Explain the expriment to identify regulatory regions
A
- Overlapping linker scanning mutations (rectangles) were performed from one end of the region under investigation to the other
- Each rectangle =position in which a linker replaced a 6-10 nucleotide segment
- Experiment shows-> thymidine kinase gene transcription is blocked by mutations in 3 distinct regions/sequence motifs that are just upstream from the transcription initiation site
4
Q
Explain what the proximal promoter
A
- CCAAT box (GGCCAATCT) +GC box (GGGCGG) are present in the promoters of many eukaryotic genes
+ are between 50 and 200 bp upstream from the transcription initiation site - The region just upstream from the core promoter =proximal promoter
- CCAAT and GC boxes are promoter proximal elements (PPEs)
5
Q
Explain what enhancers
A
- Enhancer regulatory sequences stimulate transcription
- SV40 PPE consists of six GC boxes (GGGCGG)
- Enhancer is in between 116-261 upstream of the transcriptional start site
6
Q
Explain the properties of enhancers
A
- Act at a distance
- Orientation-independent
Position can be upstream or downstream of the transcription initiation site - Can be cell-type- or tissue-specific -> active in one cell-type/tissue but NOT in another
- Activator (protein) binds to enhancer (DNA)
- Cell must have transcription activator proteins capable of binding to enhancer-> for full gene expression to occur
- Activator proteins can act in a no. of different ways to turn on gene expression
including directly interacting with the PIC + promoting open chromatin structure/euchromatin - Can be within transcription unit
7
Q
Describe how activators can act on enhancers
A
- Activator proteins can act in a no. of different ways to turn on gene expression
including directly interacting with the PIC + promoting open chromatin structure/euchromatin
8
Q
Describe what silencers and repressors are
A
- Eukaryotes have negative regulatory elements -silencers
Silencers: sequence specific DNA elements that repress transcription of a target gene - Mostly function independently of distance and orientation from/to target gene
- Transcriptional repressors: DNA binding protein that binds to silencer
9
Q
Explain how silencers can act to repress transcription
A
- Silencers act by:
- > establishing repressive chromatin /heterochromatin
- > prevent nearby transcriptional activators from binding to its binding site
- > blocking PIC formation
10
Q
List what is contained in a higher mammalian gene
A
Mammalian genes contain:
- promoter proximal elements
- enhancers
- silencers
- core promoter
11
Q
List the regulatory elements and describe how they can regulate gene expression
A
- Enhancers, silencers and PPEs consist of clusters of modules (DNA sequence motifs)
-> binds specific transcription activator or transcriptional repressor proteins
= provides a mechanism for gene regulation ->by controlling the amount of functional transcription activator or repressor proteins with the cell nucleus
12
Q
Explain how activators and depressors interact with promoter elements
A
- Transcription activator/repressor proteins have at least 2 independently folded + distinct functional domains:
DNA-binding domain -> makes sequence specific contacts with the control elements in the enhancer
Activation or repression domain is left ‘free’ -> recruits/binds various components of the transcription machinery OR to alter chromatin structure around the transcriptional start site
= to activate transcription
Additional domains include: Dimerization domains and ligand binding domains
13
Q
Describe EMSA
A
- Used to determine protein-DNA binding
- Label DNA with radioisotope + add protein or fractions of nuclear extract
- Migration of DNA fragment is slow -> when bound to protein
=causing a shift in the location of the radiolabelled DNA fragment - Visualised by autoradiography on X-ray film
14
Q
Explain how EMSA is used to identify recognition site
A
- Used to determine the exact position/sequence a protein binds to DNA
- Label DNA with radioisotope and add protein or fractions of nuclear extract
- Protein bound to DNA protects that region from digestion by a nuclease
- Region of DNA protected by the bound protein
- > the binding site appears as a gap or “footprint” in the array of bands
- Visualised by autoradiography on X-ray film after electrophoresis
15
Q
Explain how cell-based assay identifies activators
A
- Determine the type of activity of a DNA- binding protein that regulates transcription
- Activators or repressors ability to activate or repress transcription can be determined-> w/ in an in vivo transfection assay
- System requires 2 plasmids:
- > one containing the potential transcriptional activator or repressor (red)
- > other contains a reporter gene (orange) + one or more binding sites for potential activator/repressor
- Both plasmids are transfected into cells at the same time
- Production of the reporter gene mRNA (and protein) is measured
- The reporter gene -> often encodes green fluorescent protein = easy to determine if activator or repressor
- Useful for using domains/truncated proteins to identify +map activator/repressor domains
16
Q
Give the example used for identifying activators
A
- GAL4 =yeast transcriptional activator
- > binds to UAS sequences
- Use of deletion mutants-> identified both the DNA binding domain (aa 1-74) +the activation domain (aa 738-823)
- DNA binding regions are determined by EMSA
- Activation regions by cell-based reporter assay
17
Q
Explain the structure of homeoboxes and its fucntion
A
- Homeobox proteins contain a DNA-binding region called the homeodomain
- The homeodomain contains a helix-turn-helix motif
- ->highly conserved between different homeodomain-containing proteins
- The recognition helix region binds in the major groove of DNA by a.a and mediates sequence-specific binding
- Homeodomain found in transcription regulatory proteins
- Regulate many important developmental genes
18
Q
Explain the structure of zinc fingers
A
- Many eukaryotic proteins in secondary structure have regions that fold around a central Zn2+ ion -> form tertiary structure
- A pair of cysteines and a pair of histidines act together to bind a zinc ion->causing a fold known as a zinc finger
- This C2H2 zinc finger is the most common DNA-binding motif encoded in the human genome
- 23- to 26- amino acid consensus
- Many proteins 1+ ZF
19
Q
Explain the structure of zinc fingers
A
- Many eukaryotic proteins in secondary structure have regions that fold around a central Zn2+ ion -> form tertiary structure
- A pair of cysteines and a pair of histidines act together to bind a zinc ion->causing a fold known as a zinc finger
- This C2H2 zinc finger is the most common DNA-binding motif encoded in the human genome
- 23- to 26- amino acid consensus
- Many proteins 1+ ZF
20
Q
Explain the folding of proteins in their secondary structure to form zinc fingers
A
- The finger region =2 antiparallel β– sheets+ followed by an α–helix with the zinc ion buried in the interior
- Zinc ion binds with tetrahedral geometry to Cys-3 and Cys-6 in the β–strand and to His19 and His-23 in the α-helix
21
Q
Explain how proteins in zinc fingers interact with DNA/RNA and other proteins
A
- ZF present in transcription factors
- > regions outside of ZF interact with DNA/RNA/ other proteins
- 3 zinc finger protein interacts with three 3bp sub-sites on DNA as the zinc-finger regions wraps around one turn of the DNA helix
- Overall binding specificity and affinity are determined by contributions from all three zinc fingers
22
Q
Explain how hormone dependent nuclear receptors act
A
- In absence of hormone ->the nuclear receptor is kept in the cytoplasm through an interaction with inhibitors
- In presence of hormone->diffuses through plasma membrane and binds to LBD (ligand binding domain)
= releasing inhibitor - Then nuclear receptor moves to nucleus + binds as a dimer (homo- or hetero-) to response elements -> through its zinc finger DNA binding domain (DBD)
23
Q
State what parts of the nuclear receptor is conserved
A
- Highly conserved (DBD) +conserved LBD
- Activation function domains (AF1 and 2) and variable hinge region and are not conserved
24
Q
Explain how nuclear receptor domain swapping shows function
A
- Effects of swapping to have ER with DBD of GR
- Chimera causes-> binding of estrogen to ER + induces transcription of glucocorticoid responsive gene
25
Q
Explain the binding of GR to DNA
A
- GR binds to DNA as a homodimer
- Contact DNA bases in the major groove on the same side of the DNA molecule
- GR binds to glucocorticoid response element (GRE)
- GRE has two 6-bp palindromic half sites separated by three nucleotides
26
Q
Explain how leu zippers are formed
A
- Found in some proteins that contained a leucine every 7th amino acid over a 35 amino acid region
- Predicted to form an α-helix in which Leu would occur every 2 turns (α-helix has 3.6 amino acids per turn)
= Leu side chains occur on one face - α-helices segments that are either identical (homo) or different dimerise-> used to characterise proteins
- Leu side chains interdigitate like teeth on a zipper
27
Q
Explain how Leu zipper interacts with DNA
A
- The leucine zipper does NOT directly bind to DNA
- Facilitates dimerisation of the protein
- The dimerisation region often forms a coiled -coil to ‘join’ two proteins
+facilitate subsequent DNA binding - The DBD is highly basic + together with the leucine zipper forms the bzip DBD
28
Q
Give example of a protein that is leu zipper and describe how it interacts with DNA
A
- Gcn4 contains a bzip DNA binding domain
- Binds in major groove to two semi-palindromic half sites
29
Q
Give example of a proteins that can do bzip heterodimerisation and how that affects DNA binding
A
- c-Jun can bind DNA as a homodimer or as a heterodimer with c-Fos
- Dimerisation by the leucine zipper allows-> different complexes w/different binding affinities + different activity to form on an identical DNA-binding site
30
Q
State how activators with bHLH domains bind and what they contain
A
- Activators with basic- helix- loop- helix domain bind to DNA as homo- or heterodimers
- Contain a basic recognition helix and HLH dimerisation domain
31
Q
State how activators with bHLH domains bind and what they contain
A
- Activators with basic- helix- loop- helix domain bind to DNA as homo- or heterodimers
- Contain a basic recognition helix and HLH dimerisation domain
32
Q
Explain how Myc, Max and Mad proteins bind
A
- Myc and max proteins can form heterodimers
- Max and mad can form heterodimer too
- Mad is another bHLH zip protein
33
Q
Describe the structural domains and characteristics of the activators
A
- Homeobox: homeodomain-> DNA binding role-> in drosophila homeostatic genes-> DNA binding mediated through helix-turn-helix motif
- Cysteine-histidine ZF-> DNA binding role-> in TFIIIA-> multiple copies of ZF motif
- Cysteine-Cysteine ZF finger-> DNA binding role-> In nuclear receptor family-> single pair of fingers + relate motifs in GAL4 (yeast)
- Basic element-> DNA binding role-> c-fos,c-jun-> Found in association with Leu zipper
- Leu zipper-> protein dimerisation role-> c-jun, c-fos, c-myc-> mediates dimerisation =essentail for DNA binding by adjacent domain
- Helix-loop-helix-> protein dimerisation role-> c-myc->
34
Q
Describe the structural domains and characteristics of the activators
A
- Homeobox: homeodomain-> DNA binding role-> in drosophila homeostatic genes-> DNA binding mediated through helix-turn-helix motif
- Cysteine-histidine ZF-> DNA binding role-> in TFIIIA-> multiple copies of ZF motif
- Cysteine-Cysteine ZF finger-> DNA binding role-> In nuclear receptor family-> single pair of fingers + relate motifs in GAL4 (yeast)
- Basic element-> DNA binding role-> c-fos,c-jun-> Found in association with Leu zipper
- Leu zipper-> protein dimerisation role-> c-jun, c-fos, c-myc-> mediates dimerisation =essentail for DNA binding by adjacent domain
- Helix-loop-helix-> protein dimerisation role-> c-myc-> Mediates dimerisation =essentail for DNA by adjacent domain
