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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
3mRNA
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 several polypeptides, each with its own start and stop codon
The “operator” is a particular sequence of bases where the repressor can bind
DNA
mRNA
Protein
Tryptophan(corepressor)
Active repressor
No RNA made
Tryptophan present -> repressor active -> operon “off”. Figure 18.21b
Active repressor can bind to operator and block transcription
Trp operon
Lac operonInducible operons are normally off
When lactose is present, repressor can no longer bind DNA. Transcription occurs
Positive vs Negative Gene Regulation
• Both the trp and lac operons involve negative control of genes– because the operons are switched off by the active form of the
repressor protein
• Some operons are also subject to positive control– 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 food source
– When glucose is scarce, the lac operon is activated by the binding of CAP
Positive Gene Regulation- CAP
Active form of CAP helps RNA polymerase bind to promoter, so transcription can start
ATP
GTP
cAMP
Proteinkinase A
Cellular responses
G-protein-linkedreceptor
Adenylylcyclase
G protein
First messenger(signal moleculesuch as epinephrine)
You’ve seen cAMP used in other signaling pathways
•Enzyme adenylyladenylyl cyclasecyclase
• 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 cannot activate adenylate cyclase, so level of cAMP falls
Glucose transporter complex also activates adenylate cyclase
DNA binding proteins can be either repressors or activators, depending on how they intereact
with RNA polymerase
This configuration helps RNA polymerase bind
This configuration blocks RNA polymerase
Activator
Repressor
Dual control of the lac operon
off, because CAP not bound
off, because repressor active and 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 Transacetylase-Galactosidase
5
3mRNA 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?
Arabinose is another sugar that E. coli can metabolize
• Will those genes be repressible or inducible?
• How might it be regulated?
Arabinose can bind to the repressor
Arginine is an essential amino acid.
• Will that pathway be repressible or inducible?
• How might argenine synthesis be regulated?
Galactose is yet another sugar that E. coli can metabolize.
• Will those genes be repressible or inducible?
• How might gal be regulated?
O galEO galT galK
Gal repressor protein(galR)
Epimerase Transferase Kinase
P
Don’t memorize these names- just the general concept.
CAP
Galactose
Gene Regulation in Prokaryotes and Eukarykotes
• Prokaryotes– Operons
• 27% of E. coli genes• (Housekeeping genes
not in operons)
– simultaneous transcription and translation
• Eukaryotes– No operons, but they still
need to coordinate regulation
– More kinds of control elements
– RNA processing– Chromatin remodeling
• Histones must be modified to loosen DNA
Figure 19.3
Signal
NUCLEUSChromatin
Chromatin modification:
Gene
DNA Gene availablefor transcription
RNA ExonTranscription
Primary transcript
RNA 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
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