Download - Bio 101A
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Bio 101A
GE III results day
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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
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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.
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Prokaryotic Gene Regulation Bio 101A
• Operon structure and function• Positive/Negative gene regulation• Promoters, operators, repressors, inducers
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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
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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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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
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V. fischeri interacts symbiotically with a squid
The winnowing: establishing the squid–vibrio symbiosisSpencer V. Nyholm & Margaret McFall-NgaiNature Reviews Microbiology 2, 632-642 (August 2004)
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Enzyme Regulation maintains Efficiency
9
• Enzymes: General Background
• -galactosidase
• Gene Regulation
• Biochemical Regulation
• Experiment
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-galactosidase
10
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-galactosidase
11
lactose
galactose
glucose
H2O
- galactosidase(aka lactase in humans)
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-galactosidase Regulation
12
• Why Regulate - galactosidase ?
• Levels at which - galactosidase can be
regulated:
• Genetic
• Biochemical
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The lac operon regulates when β-galactosidase is made
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When lactose is present, transcription is activated
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LE 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• 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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Promoter Promoter
DNA trpR
Regulatorygene
RNApolymerase
mRNA
3
5
Protein Inactiverepressor
Tryptophan absent, repressor inactive, operon on
mRNA 5
trpE trpD trpC trpB trpA
OperatorStart codon Stop codon
trp operon
Genes of operon
E
Polypeptides that make upenzymes for tryptophan synthesis
D C B A
Prokaryotic Operon structure ensures efficient regulation of transcription
The tryptophan biosynthesis operon is repressible by the presence of its product, tryptophan
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LE 18-21b_1
DNA
Protein
Tryptophan(corepressor)
Tryptophan present, repressor active, operon off
mRNA
Activerepressor
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LE 18-21b_2
DNA
Protein
Tryptophan(corepressor)
Tryptophan present, repressor active, operon off
mRNA
Activerepressor
No RNA made
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Basic Operon Regulation
Regulator Gene
Promoter Operator Structural Genes
RNA Polymerase
Repressor mRNA
Repressor Protein
NO TRANSCRIPTION
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Tryptophan OperonTryptophan Present
Regulator Gene Promoter Operator Structural GenesAttenuator
trpR mRNA
RNA Polymerase
NO TRANSCRIPTION
TrpR protein (homodimer)
+ tryptophan (corepressor)
TrpR aporepressor + corepressor (can bind to operator)
Q: Why might the cell want to produce an aporepressor that is only activated by the operon’s end product?
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Tryptophan Operon Tryptophan Absent
Regulator Gene Promoter Operator Structural GenesAttenuator
trpR mRNA
RNA Polymerase
TRANSCRIPTION
TrpR protein (homodimer)
TrpR aporepressor(cannot bind to
operator)
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Tryptophan Repressor Protein
DNA
Tryptophan (co-repressor)
TrpR protein subunits
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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
DNA lacl
Regulatorygene
mRNA
5
3
RNApolymerase
ProteinActiverepressor
NoRNAmade
lacZ
Promoter
Operator
Lactose absent, repressor active, operon off
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LE 18-22b
DNA lacl
mRNA5
3
lac operon
Lactose present, repressor inactive, operon on
lacZ lacY lacA
RNApolymerase
mRNA 5
Protein
Allolactose(inducer)
Inactiverepressor
-Galactosidase Permease Transacetylase
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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
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 18-23b
DNA lacl
CAP-binding site
Promoter
RNApolymerasecan’t bind
Operator
lacZ
Inactive lacrepressor
InactiveCAP
Lactose present, glucose present (cAMP level low): little lacmRNA synthesized
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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.
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Operons can be cut and pasted together to make operon fusions
lacIRepressor Oper. TrpE, D, C, B, AAtt.
Tryptophan Operon Lactose OperonT Pro. Oper.
Promoter
Operator Z gene
Y gene A gene
mRNA
β-gal
Pro. 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?
lacIRepressor Oper. TrpE, D, C, B, AAtt.
Tryptophan Operon Lactose OperonT Pro. Oper.
Promoter
Operator Z gene
Y gene A gene
mRNA
β-gal
Pro. Z gene Y gene A gene
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What if we add a plasmid which contains the TrpR gene? With tryptophan? Without?
lacIRepressor Oper. TrpE, D, C, B, AAtt.
Tryptophan Operon Lactose OperonT Pro. Oper.
Promoter
Operator Z gene
Y gene A gene
mRNA
β-gal
Pro. Z gene Y gene A gene
mutant trpR-containing
plasmid
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04/22/23 VandePol 35
Manufactured by a private corporation
AraC- arabinose gene
GFP- Green Fluorescent protein
bla- Beta-lactamase ori- you know this…
pGLOori
blaGFP
araC
Another engineered plasmid with fusion Operon: pGLO
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04/22/23 36
Is this:Anabolic 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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04/22/23 38
RNA Polymerase
Z Y A
Z Y ALacI
Effector (Lactose)
Z Y ALacI
lac Operon
B A DaraC
B A DaraC
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
lac Operon and ara operon
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Grow? Glow?
Follow protocol On which plates will
colonies grow? Which colonies will
glow?
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
In eukaryotes, DNA replication begins at may sitesalong the giant DNA molecule of each chromosome.
Two daughter DNA molecules
Parental (template) strand
Daughter (new) strand0.25 µm
Replication fork
Origin of replication
Bubble
In this micrograph, three replicationbubbles are visible along the DNAof a cultured Chinese hamster cell(TEM).
DNA polymerase binds to the ori
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Gene Regulation
RNA Polymerase
araC
ara GFP Operon
GFP Gene
araC GFP Gene
araC GFP Gene
Effector (Arabinose)
B A DaraC
B A DaraC
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
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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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
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Using GFP as a biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.htmlWith permission from Marc Zimmer
GFP can be fused to cellular proteins
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GFP Fusion & Protein Localization1
2
Gene X (no stop codon)Control Region GFP gene (in-frame with Gene X)
express gene-of-interest
GFPProtein X
3Translated Fusion Protein Fluorescence Visualization
Gene Fusion
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Fusion protein in C. elegans highlights nervous system
Vesicle Transport in the Caenorhabditis elegans Nervous System
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SNB-1::GFP Fusion1
2
For snb-1Control Region GFP gene (in-frame with snb-1)
express gene-of-interest
3Neurotransmitter-Packaging Vesicles Present GFP-Tagged SNB-1 on Surface
View SNB-1 (Vesicle) Distribution
Fusion of snb-1 to GFP gene
GFPSynaptobrevin
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2008 Nobel Prize- GFP
• GFP mice
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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? Why are they there?
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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 antibiotic’s effectiveness
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What about araC?
Arabinose is a 5-carbon sugar, different from ribose