32 gene regulation, continued lecture outline 11/21/05dstratto/bcor011_handouts/32_operons2.pdf ·...
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32 Gene regulation, continued Lecture Outline 11/21/05• Review the operon concept
– Repressible operons (e.g. trp)– Inducible operons (e.g. lac)
• Positive regulation of lac (CAP)• Practice applying the operon concept to
predict:– the phenotypes of mutants– The characteristics of other operons
• Gene regulation in prokaryotes vs eukaryotes
Genes of operon
Protein
Operator
Polypeptides that make upenzymes for tryptophan synthesis
Regulatorygene
RNA polymerase
Promoter
trp operon
5′
3′mRNA
trpDtrpE trpC trpB trpAtrpRDNA
mRNA
E D C B A
The trp operon:
Figure 18.21a
5′
Tryptophan absent -> repressor inactive -> transcription
One long mRNA codes severalpolypeptides, each with its own startand stop codonThe “operator” is a
particular sequence ofbases where therepressor can bind
DNA
mRNA
ProteinTryptophan
(corepressor)
Active repressor
No RNA made
Tryptophan present -> repressor active -> operon “off”.Figure 18.21b
Active repressor canbind to operator andblock transcription
Trp operon
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Lac operonInducible operons are normally off
When lactose is present,repressor can no longerbind DNA. Transcriptionoccurs
Positive vs Negative GeneRegulation
• Both the trp and lac operons involve negativecontrol of genes– because the operons are switched off by the
active form of the repressor protein
• Some operons are also subject to positivecontrol– An activator protein is required to start
transcription.– E.g. catabolite activator protein (CAP)
Promoter
Operator
InactiveCAP
ActiveCAPcAMP
DNA
Inactive lacrepressor
lacl lacZ
Figure 18.23a
– In E. coli, glucose is always the preferred foodsource
– When glucose is scarce, the lac operon isactivated by the binding of CAP
Positive Gene Regulation- CAP
Active form ofCAP helps RNApolymerase bindto promoter, sotranscription canstart
ATP
GTP
cAMP
Proteinkinase A
Cellular responses
G-protein-linkedreceptor
AdenylylcyclaseG protein
First messenger(signal moleculesuch as epinephrine)
You’ve seencAMP used inother signalingpathways
•Enzyme adenylyladenylyl cyclasecyclase
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• When glucose is abundant,– cAMP is used up– CAP detaches from the lac operon,– prevents RNA polymerase from binding to
the promoter
Inactive lacrepressor
InactiveCAP
DNA
RNApolymerasecan’t bind
Operator
lacl lacZ
Promoter
Figure 18.23b If it is busy phosphorylating glucose, it cannotactivate adenylate cyclase, so level of cAMP falls
Glucose transporter complex also activates adenylatecyclase
How do genetic switcheswork?
DNA binding proteins can be either repressorsor activators, depending on how they intereact
with RNA polymerase
This configuration helpsRNA polymerase bind
This configuration blocksRNA polymerase
Activator
Repressor
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Dual control of the lac operon
off, because CAP not bound
off, because repressor activeand CAP not bound
off, because repressor active
Operon active
+ glucose+ lactose
+ glucose- lactose
- glucose- lactose
- glucose+ lactose
Glucose must be absent Lactose must be present
X-ray structure of CAP-cAMP bound to DNA
Many Operons use CAPlac, gal, mal, ara, etc.
CAP binds to RNA polymerase
mRNA 5'
DNA
mRNA
Protein
Allolactose(inducer)
Inactiverepressor
lacl lacz lacY lacA
RNApolymerase
Permease Transacetylaseβ-Galactosidase
5′
3′mRNA 5′
The Lac operon
Figure 18.22b What will happen if there is a deletion of the:+ lactose? - lactose?
• operator?• lac repressor gene?• CAP binding site?
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Arabinoseis another sugar that E. coli can metabolize
• Will those genes be repressible orinducible?
• How might it be regulated?
Arabinose can bind to the repressor
Arginine is an essential amino acid.
• Will that pathway be repressible orinducible?
• How might argenine synthesis beregulated?
Galactose is yet another sugarthat E. coli can metabolize.
• Will those genes be repressible orinducible?
• How might gal be regulated?
O galEO galT galK
Gal repressor protein(galR)
Epimerase Transferase Kinase
P
Don’t memorizethese names- justthe general concept.
CAP
Galactose
Gene Regulation inProkaryotes and Eukarykotes
• Prokaryotes– Operons
• 27% of E. coli genes• (Housekeeping genes
not in operons)
– simultaneoustranscription andtranslation
• Eukaryotes– No operons, but they still
need to coordinateregulation
– More kinds of controlelements
– RNA processing– Chromatin remodeling
• Histones must be modifiedto loosen DNA
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Figure 19.3
Signal
NUCLEUSChromatin
Chromatin modification:
Gene
DNA Gene availablefor transcription
RNA ExonTranscription
Primary transcriptRNA processing
Transport to cytoplasm
Intron
Cap mRNA in nucleusTail
CYTOPLASMmRNA in cytoplasm
Degradationof mRNA
Translation
PolypetideCleavage
Chemical modificationTransport to cellular
destination
Active protein
Degradation of protein
Degraded protein
Nucleosome
30 nm
(b) 30-nm fiber
DNA Packing
Protein scaffold
300 nm
(c) Looped domains (300-nm fiber)
Loops
Scaffold
700 nm
1,400 nm
(d) Metaphase chromosome
Figure 19.2
Histone Modification
Figure 19.4a
Chromatin changes
Transcription
RNA processing
mRNA degradation
Translation
Protein processingand degradation
DNAdouble helix Amino acids
availablefor chemicalmodification
Histonetails
Histone acetylation loosensDNA to allow transcription
Figure 19.4 b
Unacetylated histones Acetylated histones