Promoters
•Map ends of mRNA on DNA
•Mapping sites on DNA for protein binding
•General Properties of promoters
•Bacterial Promoters
•Promoters for eukaryotic RNA polymerases
Nuclease Protection : Map the nucleotide in DNA
that encodes the 5’ end of mRNA.
5’
3’
nontemplate
template
RNA
Hybridize RNA with a DNA probe labeled on 5’ end:
S1 nuclease (single strand specific)
Denaturing gel electrophoresis
Size = distance from labeled site to first discontinuity between
DNA and RNA, e.g. 5’ end of gene or beginning of an exon.
Primer Extension : Another method to determine
DNA sequence encoding the 5’ end of mRNA
5’
3’
nontemplate
template
RNA
Anneal a primer, complementary to RNA, labeled on 5’ end:
Reverse transcriptase + dNTPs to extend
primer to 5’ end of RNA
Denaturing gel electrophoresis
Size = distance from labeled site to the 5’ end of the mRNA
Rapid amplification of 5’ cDNA ends = 5’ RACE
5’
Reverse transcriptase
3’ CCCCC
cDNA
GGGG
5’
GGGG
CCCCC
GGGG
CCCCC
CCCCC
GGGGG
CCCCC
RNA
Copies RNA to end, adds 3-5 C’s
Anneal oligo-nt with G’ s on 3’ end
cDNA
Further extension by RT’ase of
oligo-nt template
“RACE-ready cDNA”
cDNA
Primers, Taq polymerase, dNTPs
25-35 cycles
to amplify 5’ end of cDNA by PCR
Methods for identifying the sites in DNA to
which proteins bind in a sequencespecific manner
Electrophoretic Mobility Shift Assays
DNA Footprinting Analysis
DNase Protection
Exonuclease Protection
Methylation Interference
Does a protein bind to a particular region?
(Electrophoretic Mobility Shift Assay)
• A short DNA fragment will move relatively fast in a
non-denaturing polyacrylamide gel.
• A DNA fragment bound to a protein will move
much more slowly.
• The mobility of the protein is the primary
determinant of the mobility of the protein-DNA
complex.
• Different protein-DNA complexes usually migrate
at different rates.
• Can test for sequence-specificity by adding
increasing amounts of competitor DNAs
Example of EMSA
Competitor
Extract
_
_
_
+
1
2
+
+
Sp1
E. coli
Self
+
+
+
+
+
Oct1
+
+
+
+
+
10
11
12
13
14
Complex A
Complex B
Free Probe
Lane
3
4
5
6
7
8
9
The radioactively-labeled DNA probe binds two proteins.
Each is sequence specific.
Sp1 (or something sharing its binding site) is in complex A.
Cannot determine the identity of protein in complex B from
these data.
To what specific sequence in DNA does the
protein bind ? (DNA Footprinting Assay)
• A protein bound to a specific sequence within a
DNA fragment will protect that sequence from
cleavage by DNase or chemical reagents.
• DNA outside the region of protein binding will be
sensitive to cleavage.
• After cleavage and removal of the protein, the
resulting fragments of labeled DNA are resolved
on a denaturing polyacrylamide gel.
• Protein-protected DNA results in a region with no
bands on the gel (a “footprint”); the distance from
the labeled site is determined by flanking bands.
DNase footprint
assay, part 1
Protein bound specifically
to DNA protects the DNA
from cleavage by DNase
at discrete sites.
DNase footprint assay, Part 2
Specific locations of
protected segments
show the binding site(s)
for the protein.
Example of DNase footprint analysis:
DctA bound to DNA
Purified DctA binds to two sites on DNA. Data from
Dr. Tracy Nixon.
Methylation Interference Assay determines the
nucleotides required for binding a protein
General Properties of Promoters
General features of promoters
• A promoter is the DNA sequence required
for correct initiation of transcription
• It affects the amount of product from a
gene, but does not affect the structure of
the product.
• Most promoters are at the 5’ end of the
gene.
Phenotypes of promoter mutants
• Promoters act in cis, i.e. they affect the
expression of a gene on the same
chromosome.
• Let p = promoter; lacZ is the gene encoding
beta-galactosidase.
• p+ lacZ - /p+ lacZ+: Phenotype is Lac+, i.e.
+
lacZ complements lacZ in trans.
- +
+
• p lacZ /p lacZ : Phenotype is Lac , i.e.
+
p does not complement p in trans.
Promoter alleles show cis-dominance
• The allele of the promoter that is in cis to
the active reporter gene is dominant.
• A wild-type promoter will drive expression of
a wild-type gene, but a defective promoter
will not drive expression of an wild-type
gene.
- +
• p lacZ /p lacZ +: Phenotype is Lac-, i.e.
p- cannot drive expression of lacZ+.
• p+ lacZ + /p- lacZ -: Phenotype is Lac+, i.e.
p+ can drive expression of lacZ+.
Bacterial promoters
• A combination of approaches shows that the
-10 TATAAT and -35 TTGACA sequences
are the essential DNA sequences in most E.
coli promoters
– Conservation of DNA sequences 5’ to genes
– Sequence of mutations that increase or
decrease the level of accurate transcription
– DNA sequences contacted by RNA polymerase
– Region protected from nucleases by binding of
RNA polymerase is -50 to +20.
-35 and -10 sequences
-35
16-19 bp
-10 4-8 bp +1
---TTGACA-----------TATAAT-----CAT-----AACTGT-----------ATATTA-----GTA--Unwound in open complex
Promoter mutants
Contacts with RNA
polymerase
The sigma subunit of RNA polymerase contacts both the -35
and the -10 boxes.
Alternate s factors are used to
express specific sets of genes
Factor Gene
Use
-35
Separation
-10
s70
rpoD
General
s32
rpoH
Heat Shock CCCTTGAA 13 - 15 bp
s28
fliA
Flagella
CTAAA
15 bp
GCCGATAA
rpoN
Nitrogen
starvation
CTGGNA
6 bp
TTGCA
s54
TTGACA
16 - 19 bp
TATAAT
CCCGATNT
Promoters for eukaryotic RNA
polymerases
Use of site-directed mutagenesis to define
the promoter
• Use site-directed mutations (deletions and point
mutations) in the DNA sequence to test promoter
activity.
• Ligate the mutated DNA fragments to the coding
region of a reporter gene.
– Any gene: assay for stable RNA whose 5’ end is
at the start site for transcription.
– beta-galactosidase: measure the hydrolysis of
an analog of lactose that generates a colored,
fluorescent or chemiluminescent product
– Luciferase: chemiluminescent reaction
Comparisons of promoters for eukaryotic RNA polymerases
Evidence for a Pol II promoter
RNA polymerase II promoter
Upstream binding sites
TATA box
Initiator
HBB, encodes beta-globin
Gene
TFIID binds
Conserved in many “Class II” genes
RNA polymerase I promoter
Upstream control element
-180
-107
Conserved in mammalian HBB genes
Core promoter
Directed mutation: loss of transcription Gene
UBF1 binds
UBF1 binds
Mutations cause beta-thalassemia
Specific binding of transcription
SL1 binds cooperatively with UBF1
factors
Mutation of gene encoding
RNA polymerase III promoter (5S RNA gene)
Gene
transcription
factor that binds here
Core promoter
HBB expression
+55prevents
+80
-45
+20
Promoter
for RNA Polymerase II
Comparisons of promoters for eukaryotic RNA polymerases
RNA polymerase II promoter
Upstream binding sites
TATA box
Initiator
Gene
TFIID binds
Regulate
efficiency
RNA polymerase I promoter
of utilization of
Upstream control element
minimal
promoter
-180
-107
Minimal
promoter:
Core promoter
TATA + Inr
-45
+20
Gene
UBF1 binds
UBF1 binds
SL1 binds cooperatively with UBF1
RNA polymerase III promoter (5S RNA gene)
Gene
Core promoter
Minimal promoter is needed for basal
activity and accurate initiation
• Minimal promoter is needed for the assembly of
the initiation complex at the correct site.
• TATA box
– Well-conserved sequence centered about 25 bp 5’ to
start site
– TBP and TFIID bind
• Initiator
– Short segment around start site: YANWYY, where A is
the start site
• Y = T or C, W = T or A
– Part of TFIID will bind here
Additional sequences, usually upstream,
regulate the amount of expression
• Binding sites for transcriptional regulatory proteins
are often found upstream of the minimal promoter.
• Binding of transcriptional activators will increase
the amount of transcription from the promoter
– Sp1 binds GGGGCGGGG
– CP1 binds CCAAT
• Binding and/or effects of activators can be
regulated,e.g. in response to hormones and other
signals.
• Repressors and silencing proteins decrease the
amount of transcription.
Promoter for RNA polymerase III
(5S RNA gene)
Gene
Core promoter
+55
+80
A
IIIB
C
IIIC IIIA IIIC
Binding by TFIIIn
Pol III
T FIIIB
IIIC
IIIA
IIIC
Enhancers : Additional DNA
sequences that regulate transcription
• Enhancers cause an increase in expression of a
gene.
• Can act in either orientation.
• Can act in a variety of positions:
– 5’ to gene (similar to an upstream activation sequence)
– Internal to a gene (e.g. in an intron)
– 3’ to a gene
• Can act at a considerable distance from the
gene (up to at least 50 kb in some cases).
• Contain a set of binding sites for transcriptional
activators.
Where is the 5’ end of BMB6?
0
500
1000
1500
2200
Hybridize RNA with DNA probes labeled on 5’ end:
500 bp
600 bp
Hybridize to RNA
S1 nuclease (single strand specific)
Denaturing gel electrophoresis
500 bp probe:
NO protected, labeled fragment
600 bp probe:
100 nt protected, labeled fragment
Where is the 5’ end of BMB6? Answer
0
500
1000
1500
2200
Hybridize RNA with DNA probes labeled on 5’ end:
500 bp
600 bp
Hybridize to RNA
S1 nuclease (single strand specific)
Denaturing gel electrophoresis
NO protected, labeled fragment
100 nt protected, labeled fragment
1600
Where is the promoter for BMB6?
500
0
1600
1000
A
1500
B
2200
C
D
2200
Luciferase
activity
Luciferase
100
Luciferase
100
Luciferase
50
Luciferase
Luciferase
50
0
Where is the promoter for BMB6? Answer
500
0
1600
1000
A
1500
B
2200
C
D
2200
Luciferase
activity
100
Luciferase
50
0
50
0
Luciferase
100
Luciferase
50
Luciferase
Luciferase
50
0