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Gene regulation

Control of gene expression in
prokaryotes
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Paradigm is Lactose operon
Jacob, Monad
Allows bacteria to respond to environment
Energy saving mechanism
Constitutive expression
Inducible expression

Lactose operon

The Lac repressor
Helix-turn-helix
Hinge region
Repressor binding
Multimerization domain
• 360 amino acids
• Tetrameric protein
• 10 copies per cell
• Binds to operator sequence
 35 bp palindromic sequence
 covers start of message, centered at +11
 additional sequences at –82 and + 432 (in lacZ)

• Conformational
change in protein
upon binding of
lactose

Catabolite activation

• Second layer of control
• Cells prefer glucose so use that up first
• Low glucose results in high cAMP

Catabolite activation
• cAMP binds to CRP (cAMP receptor protein)
– cAMP binding induces conformational change in CRP
• Increases CRP affinity for DNA

– CRP is a homodimer of two 210 amino acids subunits
– Contains a helix-turn-helix DNA binding motif

• CRP-cAMP binds to CRP-site on DNA
– Region -65 to -55

• Aids formation of RNA poly promoter complex
• Lactose operon is turned on more efficiently

Tryptophan operon – attenuation

Repressor-type
regulation

• Involved in the synthesis of the
amino acid tryptophan

Second control
Attenuation

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Eukaryotic gene regulation

Eukaryotic gene expression
• Selective expression in differentiated cells critical to
higher organism function
– Most pancreatic cells make digestive enzymes
– only pancreatic islet cells make insulin and glucagon

• Temporal regulation of gene expression critical to
organism development
• Basic principle the same
– Expression may be inhibited or stimulated

• Key differences
– More complex levels of regulation
– DNA more highly packaged
– Signals have to get into the nucleus

• DNA packing
– Non-expressed DNA is highly
condensed – heterochromatin
– Whereas active DNA is less
condensed – euchromatin
– Interesting case in secretory
glands of Drosophila (fruit fly)
– polytene chromosome
• Can visualize the expressing
genes in the form of
chromosome puffs

• Many factors involved in transcription
– More binding sites & elements: greater regulation

• Additional factors, additional DNA binding sites
– Enhancers – increase expression
– Silencers – decrease expression

• Help recruit RNA polymerase

How is the information teased
out of the cell?
• Experiments by William Rutter’s group
– Took the regions up-stream (5’) to the insulin
gene or the chymotrypsin gene and coupled it to
CAT (chloramphenicol acetyl transferase)
...

– Can also use this sort of genetic construction to
see what external factors will stimulate gene
expression

What do DNA binding
proteins look like

Zinc finger

• The proteins may
have multiple
domains
• One domain contains
the DNA binding
motif

Helix-loop-helix
(Helix-turn-helix)

Leucine zipper
Homeodomain

Induction type regulation

• An activator protein binds to activation site
• An inhibitory protein bound to activator protein
• e
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galactose metabolism in yeast
– Galactose present, binds to Gal80
– Gal80 falls off, activator encourages expression of
genes involved in galactose use

Activator can work through a mediator
• TATA is part of regular eukaryotic promoter,
TFIID is a transcription factor

Insulators can block activator activity
• Two layers of control

Some activators help access promoters

Repression
a
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Binds activator
c
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Demethylation
and repacking

Mix and match to give subtly
• A single factor (eg 3) can bind in concert
with a range of other factors
• The combinatorial effect allows a greater
degree of control

Co-operative binding of factors

a
...
Direct, interaction through 3rd party
c
...
Indirect, A binds close to where DNA
exits nucleosome, causes a little
unwinding, exposes B site

Silencing
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Turning off expression
Not due to repressors
Location dependent
DNA packing dependent
Yeast telomeres (very tip of DNA) is an example

Turning genes off by methylation of
DNA and histone binding

Signal transduction
• Membrane receptor
• Internal signal or messenger
• Component enters the nucleus to act as
enhancer/silencer

Receptor for cytokines

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Cytokine binds receptor
Induces dimerization
JAK kinase activated
Receptor phosphorylated
This binds STAT
STAT gets phosphorylated,
Dimerizes and moves to nucleus
Turns on gene expression

RAS pathway – cell division

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Membrane receptor for signal

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