Transcript Repressor protein

Chapter 18~Regulaton of Gene
Expression
Control of
Prokaryotic (Bacterial) Genes
2007-2008
Bacterial metabolism
• Bacteria need to respond quickly to changes in
their environment
– if they have enough of a product,
need to stop production
STOP
• why? waste of energy to produce more
• how? stop production of enzymes for synthesis
– if they find new food/energy source,
need to utilize it quickly
GO
• why? metabolism, growth, reproduction
• how? start production of enzymes for digestion
Remember Regulating Metabolism?
• Feedback inhibition
– product acts
as an allosteric inhibitor of
1st enzyme in tryptophan
pathway
– but this is wasteful
production of enzymes
Oh, I
remember this
from our
Metabolism Unit!
-
= inhibition
-
Different way to Regulate Metabolism
• Gene regulation
– instead of blocking
enzyme function, block
transcription of genes
for all enzymes in
tryptophan pathway
• saves energy by
not wasting it on
unnecessary protein
synthesis
Now, that’s a
good idea from a
lowly bacterium!
-
= inhibition
-
-
Gene regulation in bacteria
• Cells vary amount of specific enzymes by
regulating gene transcription
– turn genes on or turn genes off
STOP
GO
• turn genes OFF example
if bacterium has enough tryptophan then it doesn’t
need to make enzymes used to build tryptophan
• turn genes ON example
if bacterium encounters new sugar (energy source),
like lactose, then it needs to start making enzymes
used to digest lactose
Bacteria group genes together
• Operon
– genes grouped together with related functions
• example: all enzymes in a metabolic pathway
– promoter = RNA polymerase binding site
• single promoter controls transcription of all genes in operon
• transcribed as one unit & a single mRNA is made
– operator = DNA binding site of repressor protein
So how can these genes be turned off?
• Repressor protein
– binds to DNA at operator site
– blocking RNA polymerase
– blocks transcription
So how can these genes be turned off?
• Repressor protein
– binds to DNA at operator site
– blocking RNA polymerase
– blocks transcription
Operon model
Operon:
operator, promoter & genes they control
serve as a model for gene regulation
RNA
polymerase
RNA
repressor
TATA
polymerase
mRNA
promoter
gene1
gene2
gene3
gene4
1
2
3
4
enzyme1
enzyme2
enzyme3
enzyme4
DNA
operator
Repressor protein turns off gene by blocking
RNA polymerase binding site.
repressor
= repressor protein
Repressible operon: tryptophan
Synthesis pathway model
When excess tryptophan is present, it
binds to tryp repressor protein & triggers
repressor to bind to DNA
RNA
polymerase
– blocks (represses) transcription
RNA
TATAtrp repressor
polymerase
mRNA
promoter
gene1
gene2
gene3
gene4
1
2
3
4
enzyme1
enzyme2
enzyme3
enzyme4
DNA
trp
operator
trp
trp
trp
trp
trp
repressor
repressor protein
trp
trp
trp
conformational change in
repressor protein!
trp
repressor
tryptophan
trp
tryptophan – repressor protein
complex
Tryptophan operon
What happens when tryptophan is present?
Don’t need to make tryptophan-building enzymes
Tryptophan is allosteric regulator of repressor protein
Inducible operon: lactose
lac
lac
RNA
polymerase
lac
Digestive pathway model
lac
When lactose is present, binds to
lac repressor protein & triggers
repressor to release DNA
lac
lac
lac
RNA
TATA lac repressor
polymerase
mRNA
promoter
– induces transcription
gene1
gene2
gene3
gene4
1
2
3
4
enzyme1
enzyme2
enzyme3
enzyme4
operator
repressor
lac
conformational change in
repressor protein!
lac
repressor
repressor protein
lactose
lactose – repressor protein
complex
DNA
Lactose operon
What happens when lactose is present?
Need to make lactose-digesting enzymes
Lactose is allosteric regulator of repressor protein
1961 | 1965
Jacob & Monod: lac Operon
• Francois Jacob & Jacques Monod
– first to describe operon system
– coined the phrase “operon”
Jacques Monod
Francois Jacob
Operon summary
• Repressible operon
– usually functions in anabolic pathways
• synthesizing end products
– when end product is present in excess,
cell allocates resources to other uses
• Inducible operon
– usually functions in catabolic pathways,
• digesting nutrients to simpler molecules
– produce enzymes only when nutrient is available
• cell avoids making proteins that have nothing to do, cell
allocates resources to other uses
Positive gene control
• occurs when an activator molecule interacts directly with
the genome to switch transcription on.
• Even if the lac operon is turned on by the presence of
allolactose, the degree of transcription depends on the
concentrations of other substrates.
• The cellular metabolism is biased toward the utilization
of glucose.
Positive Gene Regulation
• Some operons are also subject to positive control
through a stimulatory protein, such as catabolite
activator protein (CAP), an activator of
transcription
• When glucose (a preferred food source of E. coli) is
scarce, CAP is activated by binding with cyclic AMP
• Activated CAP attaches to the promoter of the lac
operon and increases the affinity of RNA
polymerase, thus accelerating transcription
Positive Gene Regulation
– If glucose levels are
low (along with
overall energy levels),
then cyclic AMP
(cAMP) binds to
cAMP receptor
protein (CRP)
which activates
transcription.
• If glucose levels are
sufficient and cAMP levels
are low (lots of ATP), then
the CRP protein has an
inactive shape and cannot
bind upstream of the lac
promotor.
Control of
Eukaryotic Genes
2007-2008
The BIG Questions…
• How are genes turned on & off
in eukaryotes?
• How do cells with the same genes
differentiate to perform completely
different, specialized functions?
Evolution of gene regulation
• Prokaryotes
– single-celled
– evolved to grow & divide rapidly
– must respond quickly to changes in external
environment
• exploit transient resources
• Gene regulation
– turn genes on & off rapidly
• flexibility & reversibility
– adjust levels of enzymes
for synthesis & digestion
Evolution of gene regulation
• Eukaryotes
– multicellular
– evolved to maintain constant internal
conditions while facing changing external
conditions
• homeostasis
– regulate body as a whole
• growth & development
– long term processes
• specialization
– turn on & off large number of genes
• must coordinate the body as a whole rather than
serve the needs of individual cells
Points of control
• The control of gene expression
can occur at any step in the
pathway from gene to functional
protein
1. packing/unpacking DNA
2. transcription
3. mRNA processing
4. mRNA transport
5. translation
6. protein processing
7. protein degradation
1. DNA packing as gene control
• Degree of packing of DNA regulates transcription
– tightly wrapped around histones
• no transcription
• genes turned off
 heterochromatin
darker DNA (H) = tightly packed
 euchromatin
lighter DNA (E) = loosely packed
H
E
DNA methylation
• Methylation of DNA blocks transcription factors
– no transcription
 genes turned off
– attachment of methyl groups (–CH3) to cytosine
• C = cytosine
– nearly permanent inactivation of genes
• ex. inactivated mammalian X chromosome = Barr body
Histone acetylation

Acetylation of histones unwinds DNA

loosely wrapped around histones



enables transcription
genes turned on
attachment of acetyl groups (–COCH3) to histones


conformational change in histone proteins
transcription factors have easier access to genes
Epigenetic Inheritance
• Although the chromatin modifications just
discussed do not alter DNA sequence, they may be
passed to future generations of cells
• The inheritance of traits transmitted by
mechanisms not directly involving the nucleotide
sequence is called epigenetic inheritance
2. Transcription initiation
• Control regions on DNA
– promoter
• nearby control sequence on DNA
• binding of RNA polymerase & transcription factors
• “base” rate of transcription
– enhancer
• distant control
sequences on DNA
• binding of activator
proteins
• “enhanced” rate (high level)
of transcription
Model for Enhancer action
• Enhancer DNA sequences
– distant control sequences
• Activator proteins
– bind to enhancer sequence &
stimulates transcription
• Silencer proteins
– bind to enhancer sequence &
block gene transcription
Turning on Gene movie
Transcription complex
Activator Proteins
• regulatory proteins bind to DNA at distant
Enhancer Sites
enhancer sites
• increase the rate of transcription
regulatory sites on DNA distant
from gene
Enhancer
Activator
Activator
Activator
Coactivator
A
E
F
B
TFIID
RNA polymerase II
H
Core promoter
and initiation complex
Initiation Complex at Promoter Site binding site of RNA polymerase
Fig. 18-9-3
Promoter
Activators
DNA
Enhancer
Distal control
element
Gene
TATA
box
General
transcription
factors
DNA-bending
protein
Group of
mediator proteins
RNA
polymerase II
RNA
polymerase II
Transcription
initiation complex
RNA synthesis
3. Post-transcriptional control
• Alternative RNA splicing
– variable processing of exons creates a family of
proteins
4. Regulation of mRNA degradation
• Life span of mRNA determines amount of
protein synthesis
– mRNA can last from hours to weeks
RNA processing movie
5. Control of translation
• Block initiation of translation stage
– regulatory proteins attach to 5' end of mRNA
• prevent attachment of ribosomal subunits & initiator
tRNA
• block translation of mRNA to protein
Control of translation movie
6-7. Protein processing &
degradation
• Protein processing
– folding, cleaving, adding sugar groups,
targeting for transport
• Protein degradation
– ubiquitin tagging
– proteasome degradation
Protein processing movie
Ubiquitin
1980s | 2004
• “Death tag”
– mark unwanted proteins with a label
– 76 amino acid polypeptide, ubiquitin
– labeled proteins are broken down rapidly in
"waste disposers"
• proteasomes
Aaron Ciechanover
Israel
Avram Hershko
Israel
Irwin Rose
UC Riverside
Proteasome
• Protein-degrading “machine”
– cell’s waste disposer
– breaks down any proteins
into 7-9 amino acid fragments
• cellular recycling
play Nobel animation
Concept 18.3: Noncoding RNAs play
multiple roles in controlling gene
expression
• Only a small fraction of DNA codes for proteins,
rRNA, and tRNA
• A significant amount of the genome may be
transcribed into noncoding RNAs
• Noncoding RNAs regulate gene expression at two
points: mRNA translation and chromatin
configuration
RNA interference
• Small interfering RNAs (siRNA)
– short segments of RNA (21-28 bases) microRNA’s
• bind to mRNA
• create sections of double-stranded mRNA
• “death” tag for mRNA
– triggers degradation of mRNA
– cause gene “silencing”
• post-transcriptional control
• turns off gene = no protein produced
siRNA
Action of siRNA
dicer
enzyme
mRNA for translation
siRNA
double-stranded
miRNA + siRNA
breakdown
enzyme
(RISC)
mRNA degraded
functionally turns
gene off
6
7
Gene Regulation
protein
processing &
degradation
1 & 2. transcription
- DNA packing
- transcription factors
5
initiation of
translation
4
mRNA
processing
5. translation
- block start of
translation
2
1
initiation of
transcription
3
mRNA splicing
3 & 4. post-transcription
- mRNA processing
- splicing
- 5’ cap & poly-A tail
- breakdown by siRNA
6 & 7. post-translation
- protein processing
- protein degradation
mRNA
4 protection
Molecular Biology of Cancer
•
•
•
•
•
Oncogene
•cancer-causing genes
Proto-oncogene
•normal cellular genes
How?
1movement of DNA; chromosome
fragments that have rejoined
incorrectly
2amplification; increases the number of
copies of proto-oncogenes
3-proto-oncogene point mutation;
protein product more active or more
resistant to degradation
Tumor-suppressor genes
•changes in genes that prevent
uncontrolled cell growth (cancer growth
stimulated by the absence of
suppression)
Cancers result from a series of genetic
changes in a cell lineage
– The incidence of cancer increases with age because multiple
somatic mutations are required to produce a cancerous cell
– As in many cancers, the development of colon cancer is gradual
Turn your
Question Genes on!
2007-2008