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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. . nontemplate. 5’. template. 3’.

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promoters

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
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.

primer extension another method to determine dna sequence encoding the 5 end of mrna
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

rapid amplification of 5 cdna ends 5 race

CCCCC

cDNA

CCCCC

GGGGG

Rapid amplification of 5’ cDNA ends = 5’ RACE

5’

RNA

Reverse transcriptase

Copies RNA to end, adds 3-5 C’s

3’

cDNA

CCCCC

GGGG

Anneal oligo-nt with G’ s on 3’ end

GGGG

5’

cDNA

CCCCC

Further extension by RT’ase of

oligo-nt template

GGGG

“RACE-ready cDNA”

CCCCC

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 sequence specific manner

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

does a protein bind to a particular region electrophoretic mobility shift assay
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
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.

to what specific sequence in dna does the protein bind dna footprinting assay
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
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
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
Example of DNase footprint analysis:DctA bound to DNA

Purified DctA binds to two sites on DNA. Data from

Dr. Tracy Nixon.

general features 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
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
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
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 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

TTGACA

16 - 19 bp

TATAAT

General

s32

rpoH

CCCTTGAA

13 - 15 bp

CCCGATNT

Heat Shock

s28

fliA

CTAAA

15 bp

GCCGATAA

Flagella

Nitrogen

starvation

s54

rpoN

CTGGNA

6 bp

TTGCA

Alternate s factors are used to express specific sets of genes
use of site directed mutagenesis to define the promoter
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
evidence for a pol ii promoter
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

promoter for rna polymerase ii
Promoter for RNA Polymerase II

Regulate efficiency of utilization of minimal promoter

Minimal promoter: TATA + Inr

minimal promoter is needed for basal activity and accurate initiation
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
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.
enhancers additional dna sequences that regulate transcription
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
Where is the 5’ end of BMB6?

500

1000

1500

2200

0

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
Where is the 5’ end of BMB6? Answer

500

1000

1500

2200

0

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

1000

1500

2200

0

Where is the promoter for BMB6?

Luciferase

activity

1600

2200

A

B

C

D

100

Luciferase

100

Luciferase

50

Luciferase

50

Luciferase

0

Luciferase

where is the promoter for bmb6 answer

500

1000

1500

2200

0

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