Prokaryotic Gene Regulation Bio 101A

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Prokaryotic Gene Regulation Bio 101A

• Operon structure and function

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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

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DNA? RNA? Protein? Trait
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LE 17-3-2
DNA
TRANSCRIPTION
mRNA
Ribosome
TRANSLATION
Polypeptide
Prokaryotic cell
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transcription of DNA to mRNA starts at the
promoter, ends at the terminator
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Some important prerequisite facts

• DNA is the code to make a protein
• Some proteins are attracted to specific sequences
  of DNA
• Affinity for DNA sequences can change with
  changes in protein conformation
• A special protein (RNA polymerase) transcribes
  DNA? RNA
• Regulatory sequences of DNA dont code for any
  specific protein, but are still important

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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 squids tissue
• Bacterium has an interest in regulating
  expression of luciferase gene

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V. fischeri interacts symbiotically with a squid
The winnowing establishing the squidvibrio
symbiosis Spencer V. Nyholm Margaret
McFall-Ngai Nature Reviews Microbiology 2,
632-642 (August 2004)
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-
galactosidase
b
10
10
b
-
galactosidase
H
O
2
galactose
lactose
b
-
galactosidase
glucose
(aka lactase in humans)
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11
b
-
galactosidase
Regulation
b
Why Regulate
-
galactosidase
?

b
Levels at which
-
galactosidase
can be

regulated
Genetic

Biochemical

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LE 18-20
Regulation of enzyme production
Regulation of enzyme activity
The manufacture of enzymes responsible for the
biosynthesis of the amino acid tryptophan is
also closely regulated
Precursor
Feedback inhibition
Enzyme 1
Gene 1
Enzyme 2
Gene 2
Regulation of gene expression
Gene 3
Enzyme 3
Enzyme 4
Gene 4
Gene 5
Enzyme 5
Tryptophan
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Prokaryotic Operon structure ensures efficient
regulation of transcription
trp operon
Promoter
Promoter
Genes of operon
DNA
trpE
trpC
trpB
trpA
trpR
trpD
Operator
Stop codon
RNA polymerase
Regulatory gene
Start codon
3
mRNA 5
mRNA
5
D
B
E
C
A
Protein
Inactive repressor
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
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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

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LE 18-21b_1
DNA
mRNA
Protein
Active repressor
Tryptophan (corepressor)
Tryptophan present, repressor active, operon off
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LE 18-21b_2
DNA
No RNA made
mRNA
Protein
Active repressor
Tryptophan (corepressor)
Tryptophan present, repressor active, operon off
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Basic Operon Regulation
NO TRANSCRIPTION
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Tryptophan Operon
Tryptophan Present
Regulator Gene
Promoter
Operator
Structural Genes
Attenuator
RNA Polymerase
NO TRANSCRIPTION
trpR mRNA
Q Why might the cell want to produce an
aporepressor that is only activated by the
operons end product?
tryptophan (corepressor)
TrpR protein (homodimer)
TrpR aporepressor corepressor (can bind to
operator)
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Tryptophan Operon
Tryptophan Absent
Regulator Gene
Promoter
Operator
Structural Genes
Attenuator
RNA Polymerase
TRANSCRIPTION
trpR mRNA
TrpR protein (homodimer)
TrpR aporepressor (cannot bind to operator)
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Tryptophan Repressor Protein
TrpR protein subunits
Tryptophan (co-repressor)
DNA
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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

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LE 18-22a
Promoter
Regulatory gene
Operator
lacl
lacZ
DNA
No RNA made
3
mRNA
RNA polymerase
5
Active repressor
Protein
Lactose absent, repressor active, operon off
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LE 18-22b
lac operon
DNA
lacl
lacZ
lacY
lacA
RNA polymerase
3
mRNA
mRNA 5
5
Permease
Transacetylase
?-Galactosidase
Protein
Inactive repressor
Allolactose (inducer)
Lactose present, repressor inactive, operon on
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• 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

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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

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LE 18-23a
Promoter
DNA
lacl
lacZ
RNA polymerase can bind and transcribe
Operator
CAP-binding site
Active CAP
cAMP
Inactive lac repressor
Inactive CAP
Lactose present, glucose scarce (cAMP level
high) abundant lac mRNA synthesized
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LE 18-23b
Promoter
DNA
lacl
lacZ
CAP-binding site
Operator
RNA polymerase cant bind
Inactive CAP
Inactive lac repressor
Lactose present, glucose present (cAMP level
low) little lac mRNA synthesized
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What about the lux operon?
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Other slides I didnt 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.

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Operons can be cut and pasted together to make
operon fusions
Tryptophan Operon
Lactose Operon
lacI
Repressor
T
Pro.
Oper.
Z gene
Y gene
A gene
Oper.
TrpE, D, C, B, A
Pro.
Att.
Promoter
Operator
Z gene
Y gene
A gene
mutant trpR-containing plasmid
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If the repressor is knocked out, what will happen
in the presence of Tryptophan?
Tryptophan Operon
Lactose Operon
lacI
Repressor
T
Pro.
Oper.
Z gene
Y gene
A gene
Oper.
TrpE, D, C, B, A
Pro.
Att.
Promoter
Operator
Z gene
Y gene
A gene
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What if we add a plasmid which contains the TrpR
gene? With tryptophan? Without?
Tryptophan Operon
Lactose Operon
lacI
Repressor
T
Pro.
Oper.
Z gene
Y gene
A gene
Oper.
TrpE, D, C, B, A
Pro.
Att.
Promoter
Operator
Z gene
Y gene
A gene
mutant trpR-containing plasmid
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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

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Is thisAnabolic or Catabolic?Positive or
negative?Inducible or repressible?
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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?

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Which colonies will glow?
Grow? Glow?

• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?

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Appendix pGLO slides that may be helpful

• Stuff about GFP, arabinose, beta-lactamase, etc.

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LE 16-12
DNA polymerase binds to the ori
Parental (template) strand
0.25 µm
Origin of replication
Daughter (new) strand
Replication fork
Bubble
Two daughter DNA molecules
In this micrograph, three replication bubbles are
visible along the DNA of a cultured Chinese
hamster cell (TEM).
In eukaryotes, DNA replication begins at may
sites along the giant DNA molecule of each
chromosome.
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Gene Regulation
On pGLO, the regulatory regions of the Arabinose
operon have been glued to the structural
sequences for GFP
What will happen on the Ara () plates? What will
happen on the Ara (-) plates?
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Using GFP as a biological tracer
GFP can be fused to cellular proteins
http//www.conncoll.edu/ccacad/zimmer/GFP-ww/prash
er.html With permission from Marc Zimmer
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The pGLO plasmid

• ori- origin of replication
• GFP- green fluorescent protein
• bla- Beta-lactamase
• araC- Arabinose
• What are all the other marks?

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Beta- lactam antibiotics have a similar structure

• Includes penicillin, ampicillin, and others
• The beta-lactam ring is a square structure common
  to all

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Beta-lactamase can destroy a beta-lactam ring
Breaking the ring destroys the antibiotics
effectiveness
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What about araC?
Arabinose is a 5-carbon sugar, different from
ribose