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’

nontemplatetemplate

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’

nontemplatetemplate

RNA

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

Anneal a primer, complementary to RNA, labeled on 5’ end:

Rapid amplification of 5’ cDNA ends = 5’ RACE

5’

3’

RNA

cDNACCCCC

GGGG

Reverse transcriptase Copies RNA to end, adds 3-5 C’s

Anneal oligo-nt with G’ s on 3’ end

CCCCC cDNAGGGG

CCCCC “RACE-ready cDNA”GGGG

cDNACCCCC

CCCCCGGGGG

5’

Further extension by RT’ase of oligo-nt template

Primers, Taq polymerase, dNTPs25-35 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

Electrophoretic Mobility Shift Assays

DNA Footprinting AnalysisDNase 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

Complex A

Complex B

Free Probe

Extract

Self E. coli Sp1 Oct1Competitor

Lane

_

_

_

+ + + + + + + + + + + + +

1 2 3 4 5 6 7 8 9 10 11 12 13 14

The radioactively-labeled DNA probe binds two proteins.

Sp1 (or something sharing its binding site) is in complex A.

Cannot determine the identity of protein in complex B from these data.

Each is sequence specific.

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 specificallyto DNA protects the DNA from cleavage by DNaseat discrete sites.

DNase footprint assay, Part 2

Specific locations ofprotected 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

---TTGACA-----------TATAAT-----CAT------AACTGT-----------ATATTA-----GTA---

-35 16-19 bp -10 +14-8 bp

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 factors are used to express specific sets of genes

Factor Gene Use -35 Separation -10

General

Heat Shock

Flagella

Nitrogenstarvation

rpoD

rpoH

fliA

rpoN

TTGACA

CCCTTGAA

CTAAA

CTGGNA

16 - 19 bp

13 - 15 bp

15 bp

6 bp

TATAAT

CCCGATNT

GCCGATAA

TTGCA

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

Evidence for a Pol II promoter

Conserved in many “Class II” genes

Conserved in mammalian HBB genes

HBB, encodes beta-globin

Directed mutation: loss of trans-cription

Mutations cause beta-thalassemiaSpecific binding of transcription factors

Mutation of gene encoding transcription factor that binds hereprevents HBB expression

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 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

Gene

Core promoter+55 +80

A C

IIIA IIICIIICIIIB Binding by TFIIIn

(5S RNA gene)

IIIA IIICIIIC

TFIIIBPol III

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?

Hybridize RNA with DNA probes labeled on 5’ end:

S1 nuclease (single strand specific)

Denaturing gel electrophoresis

0 500 1000 1500 2200

600 bp500 bp

Hybridize to RNA

600 bp probe:100 nt protected, labeled fragment

500 bp probe: NO protected, labeled fragment

Where is the 5’ end of BMB6? Answer

Hybridize RNA with DNA probes labeled on 5’ end:

S1 nuclease (single strand specific)

Denaturing gel electrophoresis

0 500 1000 1500 2200

600 bp500 bp

Hybridize to RNA

100 nt protected, labeled fragmentNO protected, labeled fragment

1600

Where is the promoter for BMB6?

0 500 1000 1500 2200

1600 2200

Luciferase

Luciferase

Luciferase

Luciferase

A B C D

Luciferaseactivity

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100

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50

Luciferase 0

Where is the promoter for BMB6? Answer

0 500 1000 1500 2200

1600 2200

Luciferase

Luciferase

Luciferase

Luciferase

A B C D

Luciferaseactivity

100

100

50

50

Luciferase 0

50 0 50 0