Transcript Controling Gene Expression

Hebrews 1:1-2
1
God, who at sundry times and in
divers manners spake in time past
unto the fathers by the prophets,
2
Hath in these last days spoken unto
us by his Son, whom he hath
appointed heir of all things, by
whom also he made the worlds;
©1999 Timothy G. Standish
Controlling
Gene Expression
Timothy G. Standish, Ph. D.
©1999 Timothy G. Standish
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.

©1999 Timothy G. Standish
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

©1999 Timothy G. Standish
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
©1999 Timothy G. Standish
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 hydrolyzes
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
©1999 Timothy G. Standish
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
©1999 Timothy G. Standish
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
LacA
Repressor
No way
Jose!
Repressor
CAP
©1999 Timothy G. Standish
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
©1999 Timothy G. Standish
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
©1999 Timothy G. Standish
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
©1999 Timothy G. Standish
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
©1999 Timothy G. Standish
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
Anthranilate
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
©1999 Timothy G. Standish
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
©1999 Timothy G. Standish
The Trp Operon:
When Tryptophan Is Absent
Hey man, I’m
constitutive
RNA
RNA Operator
Repressor Promo.
Lead. Aten. trpE trpD trpC trpBPol.trpA
Pol.
Repressor
mRNA
I need
tryptophan
Repressor needs his
little buddy tryptophan if
I’m to be stopped
Repressor
©1999 Timothy G. Standish
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

©1999 Timothy G. Standish
Transcription And Translation
In Prokaryotes
5’
3’
3’
5’
RNA
Pol.
Ribosome
mRNA
Ribosome
5’
©1999 Timothy G. Standish
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
©1999 Timothy G. Standish
The mRNA Sequence Can
Fold In Two Ways
1
1
2
2
3
3
4
4
Terminator
hairpin
©1999 Timothy G. Standish
The Attenuator
When Starved For Tryptophan
5’
3’
3’
Help,
I need
Tryptophan
RNA
Pol.
2
Ribosome
5’
3
4
1
©1999 Timothy G. Standish
The Attenuator
When Tryptophan Is Present
5’
3’
3’
Ribosome
5’
2
RNA
Pol.
3
4
1
©1999 Timothy G. Standish
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

©1999 Timothy G. Standish
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!

©1999 Timothy G. Standish
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.

©1999 Timothy G. Standish
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

©1999 Timothy G. Standish
 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.
©1999 Timothy G. Standish
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
Terminator
Sequence
RNA Transcript
©1999 Timothy G. Standish
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
©1999 Timothy G. Standish
Enhancers
DNA
Many bases
5’
3’
Enhancer
5’
Promoter
TF
Transcribed Region
3’
TF
5’
TF TF RNA
RNA
Pol.
Pol.
5’
3’
RNA
©1999 Timothy G. Standish
©1999 Timothy G. Standish