Transcript Prokaryotic Gene Regulation

Prokaryotic Gene Regulation Bio
101A
• Operon structure and function
Enzymes are coded for by genes
• DNA is the code to make proteins
• Enzymes are made of protein
• In order for a cell to make an enzyme, it must
access the DNA for that enzyme
• Enzymes are very specific to their task
DNA RNA Protein Trait
LE 17-3-2
TRANSCRIPTION
DNA
mRNA
Ribosome
TRANSLATION
Polypeptide
Prokaryotic cell
transcription
of DNA to
mRNA starts at
the promoter,
ends at the
terminator
Some important prerequisite facts
1. DNA is the code to make a protein
2. Some proteins are attracted to specific
sequences of DNA
3. Affinity for DNA sequences can change with
changes in protein conformation
4. A special protein (RNA polymerase)
transcribes DNA RNA
5. Regulatory sequences of DNA don’t code for
any specific protein, but are still important
V. fischeri interacts symbiotically
with the bobtail squid
• Helps the squid camouflage
itself during nocturnal
hunting
• 95% of colonies are expelled
daily
• The rest are fed in pouches
in the squid’s tissue
• Bacterium has an interest in
regulating expression of
luciferase gene
V. fischeri interacts symbiotically
with a squid
The winnowing: establishing the squid–vibrio symbiosis
Spencer V. Nyholm & Margaret McFall-Ngai
Nature Reviews Microbiology 2, 632-642 (August 2004)
b -galactosidase
10
b-galactosidase
H 2O
galactose
lactose
b-galactosidase
(aka lactase in humans)
glucose
11
b-galactosidase Regulation
• Why Regulate
b-galactosidase
• Levels at which
regulated:
b-galactosidase
?
can be
• Genetic
• Biochemical
12
LE 18-20
The
manufacture
of enzymes
responsible
for the
biosynthesis
of the amino
acid
tryptophan is
also closely
regulated
Regulation of enzyme
activity
Precursor
Regulation of enzyme
production
Feedback
inhibition
Enzyme 1
Gene 1
Enzyme 2
Gene 2
Regulation
of gene
expression
Enzyme 3
Gene 3
Enzyme 4
Gene 4
Enzyme 5
Tryptophan
Gene 5
Prokaryotic Operon structure ensures
efficient regulation of transcription
trp operon
Promoter
Promoter
Genes of operon
DNA
Regulatory
gene
mRNA
Protein
trpE
trpR
3
RNA
polymerase
trpD
trpC
trpB
trpA
C
B
A
Operator
Start codon Stop codon
mRNA 5
5
E
Inactive
repressor
D
Polypeptides that make up
enzymes for tryptophan synthesis
Tryptophan absent, repressor inactive, operon on
The tryptophan biosynthesis operon is repressible by the
presence of its product, tryptophan
Operons: The Basic Concept
• An operon is a collection of prokaryotic genes
transcribed together on a single mRNA
transcript to serve a single purpose
• Composed of
– An operator, an “on-off” switch
– A promoter
– Genes for metabolic enzymes
• Can be switched off by a repressor protein
• A corepressor is a small molecule that binds to a
repressor to switch an operon off
LE 18-21b_1
DNA
mRNA
Active
repressor
Protein
Tryptophan
(corepressor)
Tryptophan present, repressor active, operon off
LE 18-21b_2
DNA
No RNA made
mRNA
Active
repressor
Protein
Tryptophan
(corepressor)
Tryptophan present, repressor active, operon off
Basic Operon Regulation
Repressor Protein
NO TRANSCRIPTION
RNA
Polymerase
Repressor mRNA
Regulator Gene
Promoter
Operator
Structural Genes
Tryptophan Operon
Tryptophan Present
Regulator Gene
Promoter
Operator
Attenuator
Structural Genes
RNA Polymerase
NO TRANSCRIPTION
trpR mRNA
Q: Why might the cell want
to produce an aporepressor
that is only activated by the
operon’s end product?
+ tryptophan
(corepressor)
TrpR protein
(homodimer)
TrpR aporepressor
+ corepressor (can
bind to operator)
Tryptophan Operon
Tryptophan Absent
Regulator Gene
Promoter
Operator
Attenuator
Structural Genes
RNA Polymerase
TRANSCRIPTION
trpR mRNA
TrpR protein
(homodimer)
TrpR aporepressor
(cannot bind to
operator)
Tryptophan Repressor Protein
TrpR protein
subunits
Tryptophan (corepressor)
DNA
Repressible and Inducible Operons: Two
Types of Negative Gene Regulation
• A repressible operon is one that is usually on;
binding of a repressor shuts off transcription
• The trp operon is a repressible operon
• An inducible operon is one that is usually off; a
molecule called an inducer inactivates the
repressor and turns on transcription
• The classic example of an inducible operon is
the lac operon
LE 18-22a
Promoter
Regulatory
gene
Operator
lacl
DNA
lacZ
No
RNA
made
3
mRNA
5
Protein
Lactose absent, repressor active, operon off
RNA
polymerase
Active
repressor
LE 18-22b
lac operon
DNA
lacZ
lacl
3
mRNA
5
lacA
RNA
polymerase
mRNA 5
b-Galactosidase
Protein
Allolactose
(inducer)
lacY
Inactive
repressor
Lactose present, repressor inactive, operon on
Permease
Transacetylase
• Inducible enzymes usually function in catabolic
pathways
• Repressible enzymes usually function in
anabolic pathways
• Regulation of the trp and lac operons involves
negative control of genes because operons are
switched off by the active form of the repressor
Positive Gene Regulation
• Some operons are also subject to positive
control through a stimulatory activator protein,
such as catabolite activator protein (CAP)
• When glucose (a preferred food source of E. coli
) is scarce, the lac operon is activated by the
binding of CAP
• When glucose levels increase, CAP detaches
from the lac operon, turning it off
LE 18-23a
Promoter
DNA
lacl
lacZ
CAP-binding site
Active
CAP
cAMP
Inactive
CAP
RNA
Operator
polymerase
can bind
and transcribe
Inactive lac
repressor
Lactose present, glucose scarce (cAMP level high): abundant lac
mRNA synthesized
LE 18-23b
Promoter
DNA
lacl
CAP-binding site
Inactive
CAP
lacZ
Operator
RNA
polymerase
can’t bind
Inactive lac
repressor
Lactose present, glucose present (cAMP level low): little lac
mRNA synthesized
What about the lux operon?
Other slides I didn’t talk about
• The slides following show how operons can be
cut and pasted together in novel ways.
Regulatory sequences from one operon can be
spliced to structural sequences from another,
creating a whole new input/output device.
Operons can be cut and pasted together to
make operon fusions
Tryptophan Operon
Repressor
Pro. Oper. Att.
Lactose Operon
lacI
TrpE, D, C, B, A
Promote Operator
r
Z
gene
T Pro. Oper. Z gene Y gene
Y gene
A gene
mRNA
mutant trpRcontaining
plasmid
β-gal
A gene
If the repressor is knocked out, what will happen
in the presence of Tryptophan?
Tryptophan Operon
Repressor
Pro. Oper. Att.
Lactose Operon
lacI
TrpE, D, C, B, A
Promote Operator
r
Z
gene
T Pro. Oper. Z gene Y gene
Y gene
A gene
mRNA
β-gal
A gene
What if we add a plasmid which contains the
TrpR gene? With tryptophan? Without?
Tryptophan Operon
Repressor
Pro. Oper. Att.
Lactose Operon
lacI
TrpE, D, C, B, A
Promote Operator
r
Z
gene
T Pro. Oper. Z gene Y gene
Y gene
A gene
mRNA
mutant trpRcontaining
plasmid
β-gal
A gene
Another engineered plasmid with
fusion Operon: pGLO
 Manufactured
by a
private corporation
 AraC- arabinose
gene
 GFP- Green
Fluorescent protein
 bla- Beta-lactamase
 ori- you know this…
7/7/2015
VandePol
araC
ori
pGLO
bla
GFP
33
Is this:
Anabolic or Catabolic?
Positive or negative?
Inducible or repressible?
7/7/2015
34
Expression of Green Fluorescent Protein
• How do you think this
fusion was made?
• What are the structural
sequences? The
regulatory sequences?
• What happens when
we add arabinose sugar
to these bacteria?
• What do you think is
meant by “reporter
gene”?
Grow? Glow?
Which colonies will glow?



Follow protocol
On which plates will
colonies grow?
Which colonies will
glow?
7/7/2015
36
Appendix: pGLO slides that may be
helpful
• Stuff about GFP, arabinose, beta-lactamase,
etc.
LE 16-12
DNA polymerase binds to the ori
Parental (template) strand
Origin of replication
Bubble
Daughter (new) strand
0.25 µm
Replication fork
Two daughter DNA molecules
In eukaryotes, DNA replication begins at may sites
along the giant DNA molecule of each chromosome.
In this micrograph, three replication
bubbles are visible along the DNA
of a cultured Chinese hamster cell
(TEM).
On pGLO, the regulatory regions of the
Arabinose operon have been glued to the
structural sequences for GFP
ara GFP Operon
ara Operon
B
ara
C
A D
araC
GFP Gene
Effector (Arabinose)
Effector (Arabinose)
Gene Regulation
B A D
araC
araC
GFP Gene
RNA Polymerase
RNA Polymerase
araC
B A D
araC
GFP Gene
What will happen on the Ara (+) plates?
What will happen on the Ara (-) plates?
GFP can be fused to cellular proteins
Using GFP as a biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html
With permission from Marc Zimmer
The pGLO plasmid
• ori- origin of replication
• GFP- green fluorescent
protein
• bla- Beta-lactamase
• araC- Arabinose
• What are all the other
marks?
Beta- lactam antibiotics have a similar
structure
• Includes penicillin,
ampicillin, and others
• The beta-lactam ring is
a square structure
common to all
Beta-lactamase can destroy a betalactam ring
Breaking the ring destroys the antibiotic’s effectiveness
What about araC?
Arabinose is a 5-carbon sugar, different from ribose