Transcript Foundations of Biology - Geoscience Research Institute

Ecclesiastes 3:1
1 To every thing there
is a season, and a
time to every purpose
under the heaven:
©2000 Timothy G. Standish
Initiation of
Transcription
Timothy G. Standish, Ph. D.
©2000 Timothy G. Standish
All Genes Can’t be Expressed
At The Same Time
Some gene products are needed by all cells all
the time. These constitutive genes are
expressed by all cells.
Other genes are only needed by certain cells or
at specific times, expression of these inducible
genes is tightly controlled in most cells.
For example, pancreatic b cells make insulin
by expressing the insulin gene. If neurons
expressed insulin, problems would result.
©2000 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
©2000 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. Production of lactose metabolizing
enzymes when not needed would be wasteful
Metabolizing lactose for energy only makes
sense when two criteria are met:
1 Other more readily metabolized sugar (glucose) is
unavailable
2 Lactose is available
©2000 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 - 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
©2000 Timothy G. Standish
The Lac Operon - Control
The control region is made up of two parts:
1 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
2 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
©2000 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
©2000 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
©2000 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
©2000 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
©2000 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
©2000 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
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
©2000 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
©2000 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
©2000 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
©2000 Timothy G. Standish
Transcription And Translation
In Prokaryotes
5’
3’
3’
5’
RNA
Pol.
Ribosome
mRNA
Ribosome
5’
©2000 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
©2000 Timothy G. Standish
The mRNA Sequence Can
Fold In Two Ways
1
1
2
2
3
3
4
4
Terminator
hairpin
©2000 Timothy G. Standish
The Attenuator
When Starved For Tryptophan
5’
3’
3’
Help,
I need
Tryptophan
RNA
Pol.
2
Ribosome
5’
3
4
1
©2000 Timothy G. Standish
The Attenuator
When Tryptophan Is Present
5’
3’
3’
Ribosome
5’
2
RNA
Pol.
3
4
1
©2000 Timothy G. Standish
Expression Control 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
©2000 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 x 109 base pairs or ≈ 2 m of DNA
Some gene families are located close to one
another on the same chromosome
Genes with related functions appear to be
distributed almost at random throughout the
the genome
©2000 Timothy G. Standish
Highly Packaged DNA Cannot
be Expressed
Because of its size, eukaryotic DNA must be
packaged
Heterochromatin, the most highly packaged
form of DNA, cannot be transcribed;
therefore expression of genes is prevented
Chromosome puffs on some insect
chomosomes illustrate areas of active gene
expression
©2000 Timothy G. Standish
Only a Subset of Genes is
Expressed at any Given Time
It 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.
©2000 Timothy G. Standish
Control of Gene Expression
Cytoplasm
Packaging
Degradation
DNA
Transcription
Transportation
Modification
RNA
RNA
Processing
mRNA G
G
AAAAAA
Nucleus
Export
Degradation etc.
AAAAAA
Translation
©2000 Timothy G. Standish
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
©2000 Timothy G. Standish
Three Eukaryotic
RNA Polymerases
1 RNA Polymerase I - Produces rRNA in
the nucleolus, accounts for 50 - 70 % of
transcription
2 RNA Polymerase II - Produces mRNA
in the nucleoplasm - 20 - 40 % of
transcription
3 RNA Polymerase III - Produces tRNA
in the nucleoplasm - 10 % of
transcription
©2000 Timothy G. Standish
A “Simple” Eukaryotic Gene
Transcription
Start Site
3’ Untranslated Region
5’ Untranslated Region
Introns
5’
Exon 1 Int. 1
Promoter/
Control Region
Exon 2
3’
Int. 2 Exon 3
Exons
Terminator
Sequence
RNA Transcript
©2000 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
©2000 Timothy G. Standish
Eukaryotic RNA Polymerase II
RNA polymerase is a very fancy enzyme that
does many tasks in conjunction with other
proteins
RNA polymerase II is a protein complex of
over 500 kD with more than 10 subunits:
©2000 Timothy G. Standish
Eukaryotic RNA Polymerase II
Promoters
Several sequence elements spread over about
200 bp upstream from the transcription start
site make up RNA Pol II promoters
Enhancers, in addition to promoters,
influence the expression of genes
Eukaryotic expression control involves many
more factors than control in prokaryotes
This allows much finer control of gene
expression
©2000 Timothy G. Standish
Initiation
T. F.
Promoter
T. F.
RNA
Pol. II
RNA
Pol. II
mRNA
5’
©2000 Timothy G. Standish
Eukaryotic Promoters
Promoter
5’
Exon 1
Sequence elements
TATA
~200 bp
“TATA Box”
Initiator
Transcription
start site
SSTATAAAASSSSSNNNNNNNNNNNNNNNNNYYCAYYYYYNN
(Template strand)
~-25
-1+1
S = C or G
Y = C or T
N = A, T, G or C
©2000 Timothy G. Standish
Initiation
TFIID Binding
TFIID
“TATA Box”
Transcription
start site
TBP Associated
Factors (TAFs)
-1+1
TATA Binding
Protein (TBP)
©2000 Timothy G. Standish
Initiation
TFIID Binding
Transcription
start site
TFIID
-1+1
80o Bend
©2000 Timothy G. Standish
Initiation
TFIIA and B Binding
TFIID
TFIIB
Transcription
start site
-1+1
TFIIA
©2000 Timothy G. Standish
Initiation
TFIIF and RNA Polymerase Binding
TFIID
TFIIB
Transcription
start site
-1+1
TFIIA
TFIIF
RNA Polymerase
©2000 Timothy G. Standish
Initiation
TFIIE Binding
TFIIF TFIIB
RNA Polymerase
-1+1
TFIIA
TFIIE
TFIID
Transcription
start site
TFIIE has some
helicase activity and
may by involved in
unwinding DNA so
that transcription can
start
©2000 Timothy G. Standish
Initiation
TFIIH and TFIIJ Binding
TFIIJ
TFIIH
TFIIF TFIIB
P
TFIIA
PP
RNA Polymerase
-1+1
TFIIE
TFIID
Transcription
start site
TFIIH has some
helicase activity and
may by involved in
unwinding DNA so
that transcription can
start
©2000 Timothy G. Standish
Initiation
TFIIH and TFIIJ Binding
TFIIJ
TFIIH
TFIIF TFIIB
P
PP
-1+1
TFIIE
TFIID
Transcription
start site
RNA Polymerase
TFIIA
©2000 Timothy G. Standish
Initiation
TFIIH and TFIIJ Binding
Transcription
start site
P
-1+1
PP
RNA Polymerase
©2000 Timothy G. Standish
©2000 Timothy G. Standish