Week 4-5 Notes: Control of Eukaryotic Gene Expression
PART I: Control of eukaryotic gene expression
Controlled at level of transcription initiation by opening the chromatin
Linker scanning mutagenesis determines regulatory regions in promoters
Determined by systematic replacement of short DNA segments with a DNA linker containing a random
sequence of exactly the same size
Microinjection into Xenopus oocytes allowed assaying of gene activity (amount of mRNA produced)
First used to search the promoter of the thymidine kinase gene in HSV
o Overlapping linker scanning mutations (rectangles) were performed from one end of region under
investigation to the other
o Each rectangle represents a position in which a linker replaced a 6-10 nucleotide egment
o Experiment shows that thymidine kinase gene transcription is blocked by mutations in three distinct
regions that are just upstream from initiation site
o First is the TATA box (part of core promoter for TBP), the CCAAT box & the GC box
The proximal promoter provides regulatory elements
CCAAT box (GGCCAATCT) and GC box are present in promoters of eukaryotic genes b/w 50 and 200 bp
upstream from initiation site
o Region upstream from core promoter is called the proximal promoter
CCAAT and GC boxes are promoter proximal elements
Enhancers stimulate transcription
SV40 PPE has 6 GC boxes (GGGCGG)
Enhancer: Additional regulatory sequence is b/w 116-261 upstream of transcription start site
Properties:
o Can act at a distance
o Are orientation-independent
o Position can be upstream or downstream of initiation site
o Can be cell-type or tissue-specific (i.e. enhancer is active in one cell/tissue type but no in another)
o Cell must have transcription activators capable of binding to enhancer for full gene expression
Activators can act in different ways including directly interacting with PIC and promoting
open chromatin structure (euchromatin)
Silencers block transcription
Silencers: sequence specific DNA elements that repress transcription of target gene
Function independently of distance and orientation from/to target gene
Transcriptional repressors: Binding sites for negative transcription factors
o These proteins act by establishing repressive chromatin (heterochromatin), prevent nearby
activators from binding to its binding site or by blocking PIC formation
Organisation of control elements that regulate gene expression in higher eukaryotes
Mammalian genes have core promoter (TATA box, Inr, DPE) which bind basal transcriptional machinery,
and also have PPE, enhancers & silencers
o Enhancer range in size from 50 bp to 1.5 kbp
o Enhancers, silencers & PPEs consist of cluters of modules (DNA sequence motifs) which bind specific
transcriptional activator or repressor protein
This provides mechanism for gene regulation by controlling amount of functional activator
or repressor proteins in nucleus
, Week 4-5 Notes: Control of Eukaryotic Gene Expression
Activator or repressor proteins interact with specific promoter elements
Activators/repressors have at least two independently folded and distinct functional domains:
o DNA-binding domain: makes sequence specific contacts with control elements in regulatory
promoter or enhancer
o Activation/repression domain is left ‘free’ to bind components of transcription machinery or to
alter chromatin structure around the start site in order to activate transcription
Additional domains: dimerization domains & ligand binding domains
Electrophoretic mobility shift assay (EMSA)
Used to determine protein-DNA binding
Label DNA with radioisotope and add protein of fractions of nuclear extract
EMSA of DNA fragment I reduced (retarded) when complexed to protein, causing a shift in location of the
radiolabelled DNA fragment
Visualised by autoradiography on x-ray film
Dnase I footprinting assay
Used to determine exact position/sequence a protein binds to DNA
Label DNA w/radioisotope and add protein or fractions of nuclear extract
Protein bound to DNA protects that region from digestion by a nuclease
DNA region protected by the bound protein (and therefore the binding site) appears as a gap or ‘footprint’ in
the array of bands
Visualised by autoradiography on x-ray film after electrophoresis
A cell-based assay for transcription activation by an activator protein
Determine the type of activity of a DNA-binding protein that regulates transcription
Activators/repressors can be assayed for an ability to activate or repress transcription in an in vivo
transfection assay
System requires 2 plasmids
o One contains activator or repressor (red)
o One contains a reporter gene (orange) and one or more binding sites for the protein (green)
Both plasmids transfected into cells at the same time and the production of the reporter gene mRNA and
protein is measured
o Reporter gene encodes green fluorescent protein for ease of assay
Useful for using domains/truncated proteins to identify/map activator/repressor domains
Use of deletion mutants identifies functional domains in activators
To determine functional domains in DNA-binding protein e.g. an activator/repressor domain and the
DNA-binding domain
GAL4 is a yeast transcriptional activator which binds UAS sequences
Use of deletion mutants identified DNA-binding domain (aa 1-74) and activation domain (aa 738-823)
o DNA binding regions determined by EMSA and activation regions by cell-based reporter assay
Molecular structure of transcriptional activators
Often contain more than one activation domain but usually one DNA-binding domain
Activators often grouped according to structure of their DNA-binding domains and can be classified into
numerous structural types
Homeodomain proteins have activation and DNA-binding domains
Homeodomain: DNA-binding region in homeobox proteins
o Contains a helix-turn-helix motif which is highly conserved b/w different homeodomain-containing
proteins
Recognition helix binds in the major groove of DNA and mediates sequence-specific binding
Regulate important developmental genes
The zinc finger motif
PART I: Control of eukaryotic gene expression
Controlled at level of transcription initiation by opening the chromatin
Linker scanning mutagenesis determines regulatory regions in promoters
Determined by systematic replacement of short DNA segments with a DNA linker containing a random
sequence of exactly the same size
Microinjection into Xenopus oocytes allowed assaying of gene activity (amount of mRNA produced)
First used to search the promoter of the thymidine kinase gene in HSV
o Overlapping linker scanning mutations (rectangles) were performed from one end of region under
investigation to the other
o Each rectangle represents a position in which a linker replaced a 6-10 nucleotide egment
o Experiment shows that thymidine kinase gene transcription is blocked by mutations in three distinct
regions that are just upstream from initiation site
o First is the TATA box (part of core promoter for TBP), the CCAAT box & the GC box
The proximal promoter provides regulatory elements
CCAAT box (GGCCAATCT) and GC box are present in promoters of eukaryotic genes b/w 50 and 200 bp
upstream from initiation site
o Region upstream from core promoter is called the proximal promoter
CCAAT and GC boxes are promoter proximal elements
Enhancers stimulate transcription
SV40 PPE has 6 GC boxes (GGGCGG)
Enhancer: Additional regulatory sequence is b/w 116-261 upstream of transcription start site
Properties:
o Can act at a distance
o Are orientation-independent
o Position can be upstream or downstream of initiation site
o Can be cell-type or tissue-specific (i.e. enhancer is active in one cell/tissue type but no in another)
o Cell must have transcription activators capable of binding to enhancer for full gene expression
Activators can act in different ways including directly interacting with PIC and promoting
open chromatin structure (euchromatin)
Silencers block transcription
Silencers: sequence specific DNA elements that repress transcription of target gene
Function independently of distance and orientation from/to target gene
Transcriptional repressors: Binding sites for negative transcription factors
o These proteins act by establishing repressive chromatin (heterochromatin), prevent nearby
activators from binding to its binding site or by blocking PIC formation
Organisation of control elements that regulate gene expression in higher eukaryotes
Mammalian genes have core promoter (TATA box, Inr, DPE) which bind basal transcriptional machinery,
and also have PPE, enhancers & silencers
o Enhancer range in size from 50 bp to 1.5 kbp
o Enhancers, silencers & PPEs consist of cluters of modules (DNA sequence motifs) which bind specific
transcriptional activator or repressor protein
This provides mechanism for gene regulation by controlling amount of functional activator
or repressor proteins in nucleus
, Week 4-5 Notes: Control of Eukaryotic Gene Expression
Activator or repressor proteins interact with specific promoter elements
Activators/repressors have at least two independently folded and distinct functional domains:
o DNA-binding domain: makes sequence specific contacts with control elements in regulatory
promoter or enhancer
o Activation/repression domain is left ‘free’ to bind components of transcription machinery or to
alter chromatin structure around the start site in order to activate transcription
Additional domains: dimerization domains & ligand binding domains
Electrophoretic mobility shift assay (EMSA)
Used to determine protein-DNA binding
Label DNA with radioisotope and add protein of fractions of nuclear extract
EMSA of DNA fragment I reduced (retarded) when complexed to protein, causing a shift in location of the
radiolabelled DNA fragment
Visualised by autoradiography on x-ray film
Dnase I footprinting assay
Used to determine exact position/sequence a protein binds to DNA
Label DNA w/radioisotope and add protein or fractions of nuclear extract
Protein bound to DNA protects that region from digestion by a nuclease
DNA region protected by the bound protein (and therefore the binding site) appears as a gap or ‘footprint’ in
the array of bands
Visualised by autoradiography on x-ray film after electrophoresis
A cell-based assay for transcription activation by an activator protein
Determine the type of activity of a DNA-binding protein that regulates transcription
Activators/repressors can be assayed for an ability to activate or repress transcription in an in vivo
transfection assay
System requires 2 plasmids
o One contains activator or repressor (red)
o One contains a reporter gene (orange) and one or more binding sites for the protein (green)
Both plasmids transfected into cells at the same time and the production of the reporter gene mRNA and
protein is measured
o Reporter gene encodes green fluorescent protein for ease of assay
Useful for using domains/truncated proteins to identify/map activator/repressor domains
Use of deletion mutants identifies functional domains in activators
To determine functional domains in DNA-binding protein e.g. an activator/repressor domain and the
DNA-binding domain
GAL4 is a yeast transcriptional activator which binds UAS sequences
Use of deletion mutants identified DNA-binding domain (aa 1-74) and activation domain (aa 738-823)
o DNA binding regions determined by EMSA and activation regions by cell-based reporter assay
Molecular structure of transcriptional activators
Often contain more than one activation domain but usually one DNA-binding domain
Activators often grouped according to structure of their DNA-binding domains and can be classified into
numerous structural types
Homeodomain proteins have activation and DNA-binding domains
Homeodomain: DNA-binding region in homeobox proteins
o Contains a helix-turn-helix motif which is highly conserved b/w different homeodomain-containing
proteins
Recognition helix binds in the major groove of DNA and mediates sequence-specific binding
Regulate important developmental genes
The zinc finger motif
