copyright © 2005 pearson education, inc. publishing as benjamin cummings anticipatory questions...
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Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Anticipatory Questions
• 1. What might happen if an organism had its cells expressing all genes within the genome all the time?
• 2. At what levels can control of cellular activities/pathways be controlled?
• 3. Based on our discussions up to this point, what do you think the term “negative feedback” means?
• 4. What steps are involved in the initiation of prokaryotic transcription?
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Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Learning Objectives
• understand that regulation of gene expression is a means by which to control timing and rate of generation regarding functional gene product (either RNA or polypeptide/protein).
• explain the concept of an operon in terms of components’ functions (promoter, operator, repressor, co-repressor, inducer, gene cluster, polycistronic transcript).
• compare and contrast repressible and inducible operon systems/pathways.
• compare and contrast negative versus positive regulation of operons
• apply the operon concept to gene expression as it relates to genetic engineering (specifically, our cloning and expression of the “tomato” gene).
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Individual bacteria respond to environmental change by regulating their gene expression
• A bacterium can tune its metabolism to the changing environment and food sources
• This metabolic control occurs on two levels:
– Adjusting activity of metabolic enzymes
– Regulating genes that encode metabolic enzymes
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LE 18-20LE 18-20
Regulation of enzymeactivity
Regulation of enzymeproduction
Enzyme 1
Regulation of gene expression
Enzyme 2
Enzyme 3
Enzyme 4
Enzyme 5
Gene 2
Gene 1
Gene 3
Gene 4
Gene 5
Tryptophan
Precursor
Feedbackinhibition
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Operons: The Basic Concept
• In bacteria, genes are often clustered into operons, composed of
– An operator, an “on-off” switch
– A promoter
– Genes for metabolic enzymes
• An operon can be switched off by a protein called a repressor
• A corepressor is a small molecule that cooperates with a repressor to switch an operon off
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LE 18-21aLE 18-21a
Promoter Promoter
DNA trpR
Regulatorygene
RNApolymerase
mRNA
3
5
Protein Inactiverepressor
Tryptophan absent, repressor inactive, operon on
Polycistronic* mRNA
trpE trpD trpC trpB trpA
Operator
Start codonStop codon
trp operon
Genes of operon
E
Polypeptides that make upenzymes for tryptophan synthesis
D C B A
5
* = mRNA carries the information of several genes, which are translated into several proteins
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LE 18-21b_1LE 18-21b_1
DNA
Protein
Tryptophan(corepressor)
Tryptophan present, repressor active, operon off
mRNA
Activerepressor
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LE 18-21b_2LE 18-21b_2
DNA
Protein
Tryptophan(corepressor)
Tryptophan present, repressor active, operon off
mRNA
Activerepressor
No RNA made
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Trp Operon Animation
• http://bcs.whfreeman.com/thelifewire/content/chp13/1302002.html
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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 to the operator 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, which contains genes coding for enzymes in hydrolysis and metabolism of lactose
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LE 18-22aLE 18-22a
DNA lacl
Regulatorygene
mRNA
5
3
RNApolymerase
ProteinActiverepressor
NoRNAmade
lacZ
Promoter
Operator
Lactose absent, repressor active, operon off
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LE 18-22bLE 18-22b
DNA lacl
mRNA5
3
lac operon
Lactose present, repressor inactive, operon on
lacZ lacY lacA
RNApolymerase
Polycistronic mRNA
Protein
Allolactose(inducer)
Inactiverepressor
-Galactosidase Permease Transacetylase
5
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Lac Operon Animation
http://www.sumanasinc.com/webcontent/animations/content/lacoperon.html
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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 18-23aLE 18-23a
DNA
cAMP
lacl
CAP-binding site
Promoter
ActiveCAP
InactiveCAP
RNApolymerasecan bindand transcribe
Operator
lacZ
Inactive lacrepressor
Lactose present, glucose scarce (cAMP level high): abundant lacmRNA synthesized
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LE 18-23bLE 18-23b
DNA lacl
CAP-binding site
Promoter
RNApolymerase can’t bind efficiently
Operator
lacZ
Inactive lacrepressor
InactiveCAP
Lactose present, glucose present (cAMP level low): little lacmRNA synthesized
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Catabolite Activator Protein Mechanism
• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter18/animations.html#
• Click on “combination of switches - the lac operon”
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The Arabinose Operon - A Composite of Negative & Positive Regulation
a) In the presence of arabinose:•CAP-cAMP complex and araC-arabinose complex bind to initiator region •this allows RNA polymerase to bind to the promoter •transcription begins
b) In the absence of arabinose:araC protein assumes a different conformationacts as a repressor binds to araI and a second operator region araO forms a loop this loop prevents transcription
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Regulatory gene
Operator (part of the promoter)
Promoter for the cluster of genes
B, A, and D
Application of Operons:
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araC regulatory gene
repressor
Inducer (arabinose)
Gene D
Arabinose operon with in-frame foreign DNA inserted:
Tomato gene
start startstart
startstop
stop
stop stop
Protein B Protein A Protein D
Red Fluorescent Protein (RFP)
Gene B Gene A Gene D Tomato gene
5´
3´
Polycistronic mRNA
transcription
translationtranslation
translationtranslation