Transcript Foundations of Biology - Geoscience Research Institute

Ecclesiastes 3:1, 17
1 To every thing there is a
season, and a time to every
purpose under the heaven:
17 I said in mine heart, God shall
judge the righteous and the
wicked: for there is a time
there for every purpose and for
every work.
©2000 Timothy G. Standish
Controlling
Gene Expression:
Bacteria
Timothy G. Standish, Ph. D.
©2000 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 continually expressed
genes are called constitutive genes.
 Other genes are only needed by certain cells or
at specific times. The expression of these
inducible genes is tightly controlled.
 For example, pancreas beta cells make the
protein insulin by expressing the insulin gene.
Neurons, in comparison, don’t express insulin.
©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
 E. coli is unlikely to encounter lactose, 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
©2000 Timothy G. Standish
The Lac Operon - Parts
The lac operon is made up of a control region and
four genes:
1 LacZ - b-galactosidase - Enzyme that hydrolyzes
the bond between galactose and glucose
2 LacY - Codes for a permease that lets lactose
across the cell membrane
3 LacA - Transacetylase - An enzyme whose
function in lactose metabolism is uncertain
4 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

– Promoters are specific DNA sequences to which RNA
Polymerase binds so that transcription can occur
– The lac operon promoter also has a binding site for a
protein called Catabolite Activator Protein (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
 Trp operon genes encode enzymes 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
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
©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’
Leader
peptide3’
Help,
I need
Tryptophan
RNA
Pol.
2
Ribosome
3
4
1
5’
Ribosome stalls over
sequence 1 and sequence
2 binds to sequence 3
preventing formation of
the 3, 4 hairpin
©2000 Timothy G. Standish
The Attenuator
When Tryptophan Is Present
5’
3’
Ribosome
3’
1
Leader peptide
is released
5’
2
RNA
Pol.
3 4
Ribosome passes over
sequence 1 and onto
sequence 2 allowing
sequence 3 to form the
3, 4 hairpin
©2000 Timothy G. Standish
The Attenuator
When Tryptophan Is Present
5’
3’
Ribosome
3’
1
5’
2
RNA
Pol.
3 4
The 3, 4 hairpin
destabilizes the
elongation complex
©2000 Timothy G. Standish
The Attenuator
When Tryptophan Is Present
5’
3’
Ribosome
3’
1
5’
RNA polymerase
falls off ending
transcription
2
3 4
RNA
Pol.
©2000 Timothy G. Standish
©2000 Timothy G. Standish