Transcript 32_operons2

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
The trp operon:
Tryptophan absent -> repressor inactive -> transcription
trp operon
Regulatory
gene
Promoter
Genes of operon
trpD
trpC
trpE
trpR
DNA
trpB
trpA
Operator
3
mRNA
RNA
polymerase
mRNA
5
5
E
Protein
Figure 18.21a
The “operator” is a
particular sequence of
bases where the
repressor can bind
D
C
B
A
One long mRNA codes several
Polypeptides that make up
polypeptides,
each with its own start
enzymes for tryptophan synthesis
and stop codon
Trp operon
Active repressor can
bind to operator and
block transcription
DNA
No RNA made
mRNA
Protein
Tryptophan
(corepressor)
Active
repressor
Tryptophan present -> repressor active -> operon “off”.
Figure 18.21b
Lac operon
Inducible 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)
Positive Gene Regulation- CAP
– In E. coli, glucose is always the preferred food
source
– When glucose is scarce, the lac operon is
activated by the binding of CAP
Promoter
lacl
Active form of
CAP helps RNA
polymerase bind
to promoter, so
transcription can
start
DNA
lacZ
Operator
cAMP
Inactive
CAP
Figure 18.23a
Active
CAP
Inactive lac
repressor
First messenger
(signal molecule
such as epinephrine)
Adenylyl
cyclase
G protein
G-protein-linked
receptor
GTP
ATP
cAMP
You’ve seen
cAMP used in
other signaling
pathways
•Enzyme adenylyl cyclase
Protein
kinase A
Cellular responses
• When glucose is abundant,
– cAMP is used up
– CAP detaches from the lac operon,
– prevents RNA polymerase from binding to
the promoter
Promoter
DNA
lacl
lacZ
Operator
RNA
polymerase
can’t bind
Inactive
CAP
Inactive lac
repressor
Figure 18.23b
Glucose transporter complex also activates adenylate
cyclase
If it is busy phosphorylating glucose, it cannot
activate adenylate cyclase, so level of cAMP falls
How do genetic switches
work?
DNA binding proteins can be either repressors
or activators, depending on how they intereact
with RNA polymerase
Activator
This configuration helps
RNA polymerase bind
Repressor
This configuration blocks
RNA polymerase
Dual control of the lac operon
Glucose must be absent
Lactose must be present
+ glucose
+ lactose
off, because CAP not bound
+ glucose
- lactose
off, because repressor active
and CAP not bound
- glucose
- lactose
off, because repressor active
- glucose
+ lactose
Operon active
X-ray structure of CAPcAMP bound to DNA
Many Operons use CAP
lac, gal, mal, ara, etc.
CAP binds to RNA polymerase
The Lac operon
DNA
lacl
lacz
3
mRNA
5
Figure 18.22b
lacA
RNA
polymerase
mRNA 5'
5
mRNA
-Galactosidase
Protein
Allolactose
(inducer)
lacY
Permease
Transacetylase
Inactive
repressor
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?
CAP
O
P
O
galE
Epimerase
Galactose
Gal repressor protein
(galR)
galT
galK
Transferase
Kinase
Don’t memorize
these names- just
the general concept.
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
Signal
NUCLEUS
Chromatin
Chromatin modification:
Gene available
for transcription
DNA
Gene
RNA
Transcription
Exon
Intron
RNA processing
Tail
mRNA in nucleus
Cap
Transport to cytoplasm
Degradation
of mRNA
CYTOPLASM
mRNA in cytoplasm
Translation
Polypetide
Cleavage
Chemical modification
Transport to cellular
destination
Active protein
Degradation of protein
Figure 19.3
Primary transcript
Degraded protein
DNA Packing
30 nm
Nucleosome
(b) 30-nm fiber
Protein scaffold
Loops
300 nm
(c) Looped domains (300-nm fiber)
700 nm
1,400 nm
Figure 19.2
Scaffold
Histone Modification
Chromatin changes
Transcription
RNA processing
mRNA
Translation
degradation
Protein processing
and degradation
DNA
double helix
Figure 19.4a
Histone
tails
Amino acids
available
for chemical
modification
Histone acetylation loosens
DNA to allow transcription
Unacetylated histones
Figure 19.4 b
Acetylated histones