Promoters

1
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

2
Nuclease Protection Map the nucleotide in DNA
that encodes the 5 end of mRNA.
nontemplate
5
template
3
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.
3
Nuclease Protection Map the nucleotide in DNA
that encodes the 3 end of mRNA.
nontemplate
5
template
3
RNA
Hybridize RNA with a DNA probe labeled on 3 end
S1 nuclease (single strand specific)
Denaturing gel electrophoresis
Size distance from labeled site to first
discontinuity between DNA and RNA, e.g. 3 end of
gene or end of an exon.
4
Primer Extension Another method to determine
DNA sequence encoding the 5 end of mRNA
nontemplate
5
template
3
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
5
Rapid amplification of 5 cDNA ends 5 RACE
5
RNA
Reverse transcriptase
Copies RNA to end, adds 3-5 Cs
3
cDNA
CCCCC
GGGG
Anneal oligo-nt with G s on 3 end
GGGG
5
cDNA
CCCCC
Further extension by RTase of oligo-nt template
GGGG
RACE-ready cDNA
CCCCC
Primers, Taq polymerase, dNTPs 25-35 cycles to
amplify 5 end of cDNA by PCR
6
Methods for identifying the sites in DNA to which
proteins bind in a sequence-specific manner

• Electrophoretic Mobility Shift Assays
• DNA Footprinting Analysis
• DNase Protection
• Exonuclease Protection
• Methylation Interference

7
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

8
Example of EMSA
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.
9
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.

10
DNase Footprinting Figure 3.2.5
11
DNase footprint, part 2
12
Example of DNase footprint analysisDctA bound
to DNA
Purified DctA binds to two sites on DNA. Data
from Dr. Tracy Nixon.
13
General Properties of Promoters
14
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.

15
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.

16
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.

17
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.

18
-35 and -10 sequences
-35
16-19 bp
-10
1
4-8 bp
---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.
19
Alternate s factors are used to express specific
sets of genes
20
Promoters for eukaryotic RNA polymerases
21
Use of site-directed mutagenesis to define the
promoter

• Use site-directed mutations (delns/pt 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

22
Tests for expression of the target gene

• Whole organisms
• Transgenic animals, plants or other species in
  which the DNA construct has been integrated into
  the genome
• Cell lines
• Transfected (transformed) cells (animal, plant,
  fungal, bacterial) carrying the DNA construct
  either unintegrated or integrated into the genome
• Cell-free systems
• Crude cell extracts
• Purified RNA polymerase plus GTFs plus other
  desired proteins

23
Example of mutational analysis of a eukaryotic
promoter, Fig. 3.2.8

• Decrease in RNA produced if TATA box is deleted.
  It is needed for transcription both in vivo and
  in vitro.
• Decrease in RNA produced if CCAAT box is deleted.
  It is needed for transcription in vivo.
• Linker scanning mutations (clusters of point
  mutations) reveal specific short sequences needed
  for transcription.

24
Evidence for a Pol II promoter
HBB, encodes beta-globin
Conserved in many Class II genes
Conserved in mammalian HBB genes
Directed mutation loss of trans- cription
Mutations cause beta-thalassemia
Specific binding of transcription factors
Mutation of gene encoding transcription factor
that binds here prevents HBB expression
25
Promoter for RNA Polymerase II
Regulate efficiency of utilization of minimal
promoter
Minimal promoter TATA Inr
26
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

27
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.

28
Promoter for RNA polymerase I
29
Promoter for RNA polymerase III
30
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.

31
Where is the 5 end of BMB6?
500
1000
1500
2200
0
Hybridize RNA with DNA probes labeled on 5 end
600 bp
500 bp
Hybridize to RNA
S1 nuclease (single strand specific)
Denaturing gel electrophoresis
600 bp probe 100 nt protected, labeled fragment
500 bp probe NO protected, labeled fragment
32
Where is the 5 end of BMB6? Answer
500
1000
1500
2200
0
Hybridize RNA with DNA probes labeled on 5 end
600 bp
500 bp
Hybridize to RNA
S1 nuclease (single strand specific)
Denaturing gel electrophoresis
100 nt protected, labeled fragment
NO protected, labeled fragment
1600
33
Where is the promoter for BMB6?
Luciferase activity
1600
2200
A
B
C
D
100
Luciferase
100
Luciferase
50
Luciferase
50
Luciferase
0
Luciferase
34
Where is the promoter for BMB6? Answer
Luciferase activity
1600
2200
A
B
C
D
100
Luciferase
0
50
50
0
100
Luciferase
50
Luciferase
50
Luciferase
0
Luciferase