Transcript Prokaryotic Gene Regulation

Bio 101A
GE III results day
Which one of the following occurs when RNA
polymerase attaches to the promoter DNA?
A) elongation of the growing RNA molecule
B) termination of the RNA molecule
C) addition of nucleotides to the DNA template
D) initiation of a new RNA molecule
E) initiation of a new polypeptide chain
Which of the following is the enzyme that HIV
uses to synthesize DNA on an RNA template?
A) ligase
B) RNA polymerase
C) terminator enzyme
D) reverse transcriptase
E) None of the choices are correct.
Prokaryotic Gene Regulation Bio
101A
• Operon structure and function
• Positive/Negative gene regulation
• Promoters, operators, repressors, inducers
Q: What is “regulation”?
"When I was warning about the danger ahead on Wall Street months ago because of
the lack of oversight, Senator McCain was telling the Wall Street Journal -- and I
quote -- 'I'm always for less regulation.' " – Sen. Barack Obama
“Senator Obama was silent on the regulation of Fannie Mae and Freddie Mac, and
his Democratic allies in Congress opposed every effort to rein them in…last year he
said that subprime loans had been, quote, “a good idea.””- Sen. John McCain
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
V. fischeri interacts symbiotically
with a 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)
Enzyme Regulation maintains Efficiency
• Enzymes: General Background
• b-galactosidase
• Gene Regulation
• Biochemical Regulation
• Experiment
9
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
The lac operon regulates when βgalactosidase is made
When lactose is present,
transcription is activated
LE 18-20
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
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
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
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
In a prokaryote, a group of genes with related
functions, along with their associated control
sequences, defines
A) an allele.
B) an operon.
C) a locus.
D) a transposon.
E) a chromosome.
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…
7/15/2015
VandePol
araC
ori
pGLO
bla
GFP
35
Is this:
Anabolic or Catabolic?
Positive or negative?
Inducible or repressible?
7/15/2015
36
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”?
lac Operon and ara operon
lac Operon
LacI
Z
Y
ara Operon
A
araC
B
A
D
Effector
(Lactose)
LacI
Z
Y
A
Effector
(Arabinose)
araC
B
A
RNA Polymerase
Z
7/15/2015
Y
A
D
RNA Polymerase
araC
B
A
D
38
Grow? Glow?
Which colonies will glow?



Follow protocol
On which plates will
colonies grow?
Which colonies will
glow?
7/15/2015
39
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?
Green Fluorescent Protein
•
discovered in 1960s by Dr. Frank
Johnson and colleagues
•
closely related to jellyfish aequorin
•
absorption max = 470nm
•
emission max = 508nm
•
238 amino acids, 27kDa
•
“beta can” conformation: 11
antiparallel beta sheets, 4 alpha
helices, and a centered chromophore
•
amino acid substitutions result in
several variants, including YFP, BFP,
and CFP
40 Å
30 Å
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
GFP Fusion & Protein Localization
1
Gene Fusion
Control Region Gene X (no stop codon)
GFP gene (in-frame with Gene X)
express gene-of-interest
2
Translated Fusion Protein
Protein X GFP
3
Fluorescence Visualization
Fusion protein in C. elegans highlights nervous
system
Vesicle Transport in the Caenorhabditis elegans
Nervous System
SNB-1::GFP Fusion
1
Fusion of snb-1 to GFP gene
Control Region For snb-1
GFP gene (in-frame with snb-1)
express gene-of-interest
2
Neurotransmitter-Packaging Vesicles
Present GFP-Tagged SNB-1 on
Surface
SynaptobrevinGFP
3
View SNB-1 (Vesicle) Distribution
2008 Nobel Prize- GFP
• GFP mice
The pGLO plasmid
• ori- origin of replication
• GFP- green fluorescent
protein
• bla- Beta-lactamase
• araC- Arabinose
• What are all the other
marks? Why are they
there?
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