Regulatory mutations and eukaryotic gene regulation

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Lac operon

Analyzing phenotypes in which gene regulation is disrupted

genotype -IPTG +IPTG interpretation

I+ Z+ - + normal

I- Z+ + + constitutive

I+ Z- - - uninducible

I- Z+ / F’ I+ Z+ - + I- is recessive

B-gal activity

Notation“F’ ABC“: Addition of separate strand of DNA that replicates within bacterial host (called a plasmid) that contains the ABC genes

Bacteria are haploid, but giving cells genes on the plasmid allows determination of the consequence of allele combinations

The “cis-versus-trans” test:I acts in trans

genotype -IPTG +IPTG interpretation

I+ Z+ - + normal

I- Z+ + + constitutive

I+ Z- - - uninducible

I- Z+ / F’ I+ Z+ - + I- is recessive

I+ Z-/ F’ I- Z+ - + I acts in trans

I- Z-/ F’ I+ Z+ - + I acts in trans

B-gal activity

The “cis-versus-trans” test:O acts in cis

genotype -IPTG +IPTG interpretation

O+ Z+ - + normal

O- Z+ + + constitutive

O+ Z- - - uninducible

O- Z+/ F’ O+

Z+

+ + O- is dominant

O- Z+/ F’ O+ Z- + +

O- Z-/ F’ O+

Z+

- +

B-gal activity

O acts in cis

Maltose operon

genotype -maltose +maltose

1 Q+ T+ - +

2 Q+ T- - -

3 Q- T+/ F’ Q+ T- - +

4 Q- T-/ F’ Q+ T+ - +

5 Q+ M+ - +

6 Q+ M- - -

7 Q- M+/ F’ Q+ M- - -

8 Q- M-/ F’ Q+ M+ - +

Interpretations?A. T acts in transB. T acts in cisC. M acts in transD. M acts in cis

What can be concluded?1. A and C2. B and C3. A and D4. B and D

Q activity

Maltose operon

genotype -maltose +maltose

1 Q+ T+ - +

2 Q+ T- - -

3 Q- T+/ F’ Q+ T- - +

4 Q- T-/ F’ Q+ T+ - +

5 Q+ M+ - +

6 Q+ M- - -

7 Q- M+/ F’ Q+ M- - -

8 Q- M-/ F’ Q+ M+ - +

What can be concluded?A. T is an activatorB. T is a repressorC. Not enough information

Q activity

I made a mistake in discussing this slide. The correct answer is A,because when T is missing there is no expression of Q. Therefore, T must be acting as an activator.

If T is a trans-acting activator, thenmaltose must induce T binding to DNA

M is a cis-acting sequence

Promoter QT

RNA polMaltose

Transcription of Q

M

Prediction: If T is missing no expression of Q becauseRNApol is not recruited to the poor promoter element

If T is a trans-acting Repressor, thenmaltose prevents T binding to DNA

M is a cis-acting sequence

Promoter QT

RNA pol

Maltose

Transcription of Q

M

Maltose

+ Prediction: If T is missingRNApol recruitment tothe promoter will not beblocked and Q expressionwill be constitutive.

Minimal requirements for specific gene regulation

1. An activity that can promote or inhibit gene expression upon a stimulus– Resides in a molecule that acts in trans

2. A way to target that activity to a particular gene – DNA/RNA sequences that act in cis

Essentially any step in gene expression can be regulated

Estrogen receptor

ERE

ERRNA pol

Estrogen

Nucleus

cytoplasm

ER

ER ER

Outside of cell

ER ER

MediatorCyclin D protein

Triggers S phase and cell proliferation

cyclinDTATA

TBP

Estrogen binding changes 3-D shape of ER protein, allowing it to dimerize

Estrogen receptor transactivation but not DNA binding is inhibited by tamoxifen

ERE

ER

Estrogen

Nucleus

cytoplasm

ER

ER ER

Outside of cell

ER ER

Tumor cell fails to proliferate

cyclinDTATA

tamoxifen

Sequence-specific RNA binding proteins can modulate post-transcriptional events such as splicing

sam68rasCancerous state

ex1 ex2 ex3 ex4 ex5 ex6 ex7 ex8 ex9

cap AAAAAAex1 ex2 ex3 ex4 ex5 ex6 ex7 ex8 ex9

cap AAAAAA1 2 3 4 5 6 7 8 9

CD44 gene

CD44 Exon5 inclusion: cancer cell migration (metastasis)

Translation can be controlled by sequence-specific RNA binding proteins

BRE

bicoid

4Ecap

AAAAAA

Ribosome can’t bind mRNA

AUG

STOP

caudal

caudalbicoid

Fly head

How to study regulation? Finding regulatory sequences

Luciferase

Luciferase

Luciferase

Luciferase

Luciferase

Luciferase

100%

Luciferase

100%

100%

1%

1%

100%

1%

Luciferase activity

Recombinant DNA: regulatory DNA region + reporter

Detecting mRNA: Northern blot• Use labeled antisense DNA or RNA, hybridizes

to mRNA

Detecting protein: Western blot• Use antibody, binds specific protein