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Controlling Procaryotic
Transcription
Lecture 12
Differential Gene Expression
• Some proteins present at 100,000 copies per cell
– Others only at <10 copies per cell
• Consequence of strong and weak promoters
– Close to consensus sequence tend to be transcribed
more frequently
• Every 2 seconds
– Poorer fits for sigma give weaker promoters
• Changes in the TTGACA or the TATAAT or the spacing
between them
• Only initiated once every 10 mins
• Constitutive vs Inducible/Repressible
– Expressed at a constant number per cell at all times
• “Housekeeping genes”
– Others are turned on/off at different times
Contributing factors
• Cis factors only affect one gene
– local
– Eg, DNA sequences around the promoter
• Trans factors affect many genes
– global
– proteins in addition to RNA pol
• DNA binding activators – recruit RNA pol
• Repressors which prevent RNA pol from binding
• Also called TRANSCRIPTION FACTORS
Activators
• Activating accessory proteins can bind near the
promoter
– Eg, CAP (catabolite activator protein) binds at -80 of
several genes
– Interacts with alpha subunit of RNA pol and brings
RNA pol into the promoter
• In bugs these sequences are called Proximal
Promoter Elements
– In eucaryotes, the regulatory sequences can be miles
away!
• Called enhancer sequences
• Remember the hugely coiled nature of eucaryotic DNA
Repressors
• Repressing proteins can also bind near
the promoter
– Often close to the start (eg, -10)
– So they block the RNA pol
• Balance between activators and
repressors determines the rate of
transcription
Modulating the Factors
• The binding affinity of the activators and
repressors for DNA can change
– As can their ability to activate or block RNA pol
– Most often change is through binding smaller
molecules
• Classic examples
– The lac system codes for the genes necessary for
lactose breakdown
• Normally bugs don’t encounter lactose but, if it’s around, it
makes sense to make the necessary enzymes!
– The trp system codes for the genes necessary for
tryptophan synthesis
• Normally bugs need to make this amino acid but, if it’s
around, it makes sense not to bother making all the enzymes
Keeping lac genes repressed
Normally bugs do not make the enzymes necessary for lactose metabolism
A repressor protein is continually
(constitutively) made
repressor
Promoter region
The repressor binds to
a sequence in front of
the normal promoter
and blocks RNA pol
Genes for lactose metabolism
Lactose Derepression
Lactose arrives
The repressor has a binding
site for lactose. When
lactose is bound, the
repressor no longer binds to
the DNA.
repressor
RNA pol
Promoter region
Genes for lactose metabolism
RNA pol is free to transcribe the gene.
Activating lac genes
But the promoter is weak (quite different from the consensus
sequence). RNA pol needs a hand to get going. The binding of CAP
upstream is needed.
CAP
RNA pol
Promoter region
Genes for lactose metabolism
But CAP won’t bind if the cell is already using a better fuel (eg, glucose).
How does CAP know whether it should be binding or not?
Expression of lac genes
In order to bind to the promoter, CAP needs to binds a molecule called
cyclic AMP (cAMP)
cAMP is low when the cells are using glucose but is high when the
cells are using inferior fuels.
RNA pol
cAMP
CAP
Promoter region
Genes for lactose metabolism
When cAMP is bound to CAP, RNA pol is able to transcribe the lac genes
The interaction is between the CAP and the alpha subunit of RNA pol
Control of Biosynthetic Genes
The repressor doesn’t
bind to the promoter, so
RNA pol is free to do
it’s job
The repressor is produced continually
(constitutively) at low level
repressor
RNA pol
Promoter region
Genes for amino acid production
The promoter is nice and strong… so transcription occurs easily.
The default position is that bugs want to make the enzymes necessary for
creating the amino acids
Control of Biosynthetic Genes
Amino acid (trp)
When the amino acids
are around, they bind
to the repressor
Promoter region
The repressor now binds
to the DNA and blocks
RNA pol.
Genes for amino acid production
When there is plenty of the amino acid around, there’s no point in
transcribing the genes for its synthesis.
General Themes
• Genes coding for degradative enzymes
– Normally repressed unless they are needed
• Genes coding for biosynthetic enzymes
– Normally expressed unless there’s plenty of the product around
• Note how the affinity of the protein for DNA changes
several thousand fold by binding the compound
• A protein exerts POSITIVE control
– When its binding causes an event
– Eg, activator like CAP
• A protein exerts NEGATIVE control
– When its binding prevents an event
– Eg, repressor
• A gene is never totally off (except in spores!)
Changing Large Groups of Genes
• One type of protein can affect many genes
– CAP has a binding site on many genes
– So several genes are switched on when it is activated
• For polycistronic mRNAs, many genes affected by one promoter
– Groupings of several genes behind one promoter are called OPERONS
• Trp operon and lac operon
• Sometimes whole panels of genes need to be switched on
– Nitrogen starvation genes have CTGGNAN6TTGCA promoters
• When faced with N-starvation a special sigma (σ60) is produced that
recognises the specific promoters
– Heat shock genes have CNCTTGAAN14CCCATNT promoters
• Recognised by σ32 which, itself, if produced when the temperature changes
– Bacteriophages (viruses which invade bugs) produce their own sigma
proteins
• Allow their own genes to be expressed in preference to their hosts
Mutations in Control Processes
• Classic exam question
• What happens when there’s a mutation?
– In the promoter sequence?
– In the proximal promoter or repressor binding
sites?
– In the activator or repressor proteins?
– In the gene products?
Text Book Refs
• Chapter 11
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p223 – opening paragraphs on why gene regulation is so exciting
p224 - principles explained pretty well
p225 – positive and negative regulation explained
DO NOT FRET OVER p226-229 on the lac operon – it is too detailed
But on p230, the numbered points are manageable
Fig 11-6 is OK
p232 – the stuff on CAP (called CRP in your book) is too detailed.. but
read it anyway 
p236 on the trp operon is a bit hard but the first paragraph on p237
gives the principles pretty well
p240 – is good on the differences between global and specific
p241 – the section on constitutive, housekeeping genes
NB: I don’t think there’s any references in the book to the special sigma
subunits used to regulate heat-shock, N-starvation or bacteriophage
expression.