Download Controling Gene Expression

Controlling
Gene Expression
Timothy G. Standish, Ph. D.
All Genes Can’t be Expressed
At The Same Time
Some genes are needed for the function of all
cells all the time. These genes are called
constitutive genes and are expressed by all cells.
 Other genes are only needed by certain cells or at
specific times. The expression of these inducible
genes is tightly controlled in most cells.
 For example, beta cells in the pancreas make the
protein insulin by expressing the insulin gene. If
neurons expressed insulin, problems would result.

Operons Are Groups Of Genes
Expressed By Prokaryotes
The genes grouped in an operon are all
needed to complete a given task
 Each operon is controlled by a single
control sequence in the DNA
 Because the genes are grouped together,
they can be transcribed together then
translated together

The Lac Operon
Genes in the lac operon allow E. coli bacteria to
metabolize lactose
 Lactose is a sugar that E. coli is unlikely to
encounter, so it would be wasteful to produce the
proteins needed to metabolize it unless necessary
 Metabolizing lactose for energy only makes sense
when two criteria are met:

– Other more readily metabolized sugar (glucose) is
unavailable
– Lactose is available
The Lac Operon - Parts
The lac operon is made up of a control region and
four genes
 The four genes are:

– LacZ - b-galactosidase - An enzyme that hydrolizes
the bond between galactose and glucose
– LacY - Codes for a permease that lets lactose across
the cell membrane
– LacA - Transacetylase - An enzyme whose function in
lactose metabolism is uncertain
– Repressor - A protein that works with the control
region to control expression of the operon
The Lac Operon - Control
The control region is made up of two parts:
 Promoter

– These are specific DNA sequences to which RNA
Polymerase binds so that transcription can occur
– The lac operon promoter also has a binding site for
another protein called CAP

Operator
– The binding site of the repressor protein
– The operator is located downstream (in the 3’
direction) from the promoter so that if repressor is
bound RNA Polymerase can’t transcribe
The Lac Operon:
When Glucose Is Present But Not Lactose
Come on,
let me through
Hey man, I’m
constitutive
Repressor
CAP
Binding
Repressor
mRNA
RNA
Pol.
Promoter Operator
LacZ
LacY
Repressor
No way
Jose!
Repressor
CAP
LacA
The Lac Operon:
When Glucose And Lactose Are Present
Great, I can
transcribe!
Hey man, I’m
constitutive
Repressor
CAP
Binding
RNA
Pol.
Promoter Operator
X
Repressor
mRNA
Repressor
Repressor
LacZ
LacY
RNA
LacA
Pol.
Repressor
This lactose has
bent me
out of shape
CAP
Some transcription
occurs, but at a slow rate
The Lac Operon:
When Lactose Is Present But Not Glucose
Hey man, I’m
constitutive
Repressor
CAP
Binding
CAP
Bind to me
Polymerase
Yipee…!
RNA
Pol.
Promoter Operator
cAMP
X
Repressor
mRNA
LacZ
RNA
LacA
Pol.
LacY
Repressor
CAP
cAMP
Repressor
Repressor
This lactose has
bent me
out of shape
cAMP
CAP
The Lac Operon:
When Neither Lactose Nor Glucose Is Present
Hey man, I’m
constitutive
Repressor
CAP
Binding
CAP
Bind to me
Polymerase
RNA
Pol.
Alright, I’m off to
the races . . .
Come on, let
me through!
Promoter Operator
LacZ
LacY
LacA
Repressor
cAMP
Repressor
mRNA
Repressor
STOP
Right there
Polymerase
CAP
cAMP
cAMP
CAP
The Trp Operon
Genes in the trp operon allow E. coli bacteria to
make the amino acid tryptophan
 Enzymes encoded by genes in the trp operon
are all involved in the biochemical pathway that
converts the precursor chorismate to
tryptophan.
 The trp operon is controlled in two ways:

– Using a repressor that works in exactly the opposite
way from the lac operon repressor
– Using a special attenuator sequence
The Tryptophan
Biochemical Pathway
COO-
Glutamine Glutamate +
Pyruvate
COO-
CH2
5-Phosphoribosyla-Pyrophosphate
NH2
COO-
HO H
O C
H Anthranilate synthetase
(trpE and D)
Chorismate
OH OH
-2O PO
3
CH2 C
C
C
-OOC
OH
PPi
Anthranilate synthetase
-2O P
3
O
CH2
Antrhanilate
H
CH2 C
N-(5’-Phosphoribosyl)Anthranilate isomerase Indole- H
Enol-1-oH H C
3’-glycerol phosphate synthetase
N
Carboxyphenylamino H H
-1-deoxyribulose phosphate Glyceraldehyde- Tryptophan synthetase
(trpB and A)
H
3-phosphate
Serine
H2O
-OOC
C
C
HN
N-(5’H Phosphoribosyl)
-anthranilate
OH
H
H
N-(5’-Phosphoribosyl)-anthranilate OH
isomerase Indole-3’-glycerol
OH OH
phosphate synthetase (trpC)
CO2+H2O
-2O PO
3
O
-OOC
C
H
C
H N
H
Indole-3-glycerol phosphate
CH2
NH3+
Tryptophan synthetase
N
H
Indole
N
H
Tryptophan
The Trp Operon:
When Tryptophan Is Present
Hey man, I’m
constitutive
Repressor
RNA
Pol.
Foiled
Again!
Promo. Operator Lead. Aten. trpE trpD trpC trpB trpA
Repressor
Trp
Repressor
mRNA
STOP
Right there
Polymerase
Repressor
Trp
Attenuation
The trp operon is controlled both by a
repressor and attenuation
 Attenuation is a mechanism that works only
because of the way transcription and
translation are coupled in prokaryotes
 Therefore, to understand attenuation, it is
first necessary to understand transcription
and translation in prokaryotes

Transcription And Translation
In Prokaryotes
5’
3’
3’
5’
RNA
Pol.
Ribosome
mRNA
5’
Ribosome
The Trp Leader and
Attenuator
Met-Lys-Ala-Ile-Phe-ValAAGUUCACGUAAAAAGGGUAUCGACA-AUG-AAA-GCA-AUU-UUC-GUALeu-Lys-Gly-Trp-Trp-Arg-Thr-Ser-STOP
CUG-AAA-GGU-UGG-UGG-CGC-ACU-UCC-UGA-AACGGGCAGUGUAUU
1
2
CACCAUGCGUAAAGCAAUCAGAUACCCAGCCCGCCUAAUGAGCGGGCUUUU
3
4
Met-Gln-Thr-Gln-Lys-Pro
UUUU-GAACAAAAUUAGAGAAUAACA-AUG-CAA-ACA-CAA-AAA-CCG
trpE . . .
Terminator
The mRNA Sequence Can
Fold In Two Ways
1
1
2
2
3
3
4
4
Terminator
hairpin
The Attenuator
When Starved For Tryptophan
5’
3’
3’
Help,
I need
Tryptophan
RNA
Pol.
2
Ribosome
3
4
1
5’
The Attenuator
When Tryptophan Is Present
5’
3’
3’
Ribosome
5’
2
RNA
Pol.
3
4
1
Control Of Expression In
Eukaryotes
Some of the general methods used to control
expression in prokaryotes are used in eukaryotes,
but nothing resembling operons is known
 Eukaryotic genes are controlled individually and
each gene has specific control sequences
preceding the transcription start site
 In addition to controlling transcription, there are
additional ways in which expression can be
controlled in eukaryotes

Eukaryotes Have Large
Complex Genomes
The human genome is about 3 x 109 base
pairs or ≈ 1 m of DNA
 Because humans are diploid, each nucleus
contains 6 3 x 109 base pairs or ≈ 2 m of
DNA
 That is a lot to pack into a little nucleus!

Eukaryotic DNA Must be
Packaged
Eukaryotic DNA exhibits many levels of
packaging
 The fundamental unit is the nucleosome,
DNA wound around histone proteins
 Nucleosomes arrange themselves together
to form higher and higher levels of
packaging.

Highly Packaged DNA Cannot
be Expressed
The most highly packaged form of DNA is
“heterochromatin”
 Heterochromatin cannot be transcribed,
therefore expression of genes is prevented
 Chromosome puffs on some insect
chomosomes illustrate where active gene
expression is going on

 It
Only a Subset of Genes is
Expressed at any Given Time
takes lots of energy to express genes
 Thus it would be wasteful to express all
genes all the time
 By differential expression of genes, cells
can respond to changes in the environment
 Differential expression allows cells to
specialize in multicelled organisms.
 Differential expression also allows
organisms to develop over time.
Control of Gene Expression
Cytoplasm
Packaging
Degradation
DNA
Transcription
Transportation
Modification
RNA
RNA
Processing
mRNA G
G
AAAAAA
Nucleus
Export
Degradation etc.
AAAAAA
Translation
Logical Expression Control Points
Increasing cost
DNA packaging
 Transcription
 RNA processing
 mRNA export
 mRNA masking/unmasking
and/or modification
 mRNA degradation
 Translation
 Protein modification
 Protein transport
 Protein degradation

The logical
place to
control
expression is
before the
gene is
transcribed
A “Simple” Eukaryotic Gene
Transcription
Start Site
5’
5’ Untranslated Region
Introns
Exon 1 Int. 1
Promoter/
Control Region
3’ Untranslated Region
Exon 2
3’
Int. 2 Exon 3
Exons
RNA Transcript
Terminator
Sequence
Enhancers
DNA
Many bases
5’
3’
Enhancer
5’
Promoter
TF
Transcribed Region
3’
TF
5’
TF TF RNA
RNA
Pol.
Pol.
5’
RNA
3’
Eukaryotic mRNA
5’ Untranslated Region
5’ G
Exon 1 Exon 2
3’ Untranslated Region
Exon 3
AAAAA
3’
Protein Coding Region
5’ Cap

RNA processing achieves three things:




3’ Poly A Tail
Removal of introns
Addition of a 5’ cap
Addition of a 3’ tail
This signals the mRNA is ready to move out
of the nucleus and may control its lifespan
in the cytoplasm