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Alberts • Johnson • Lewis • Raff • Roberts • Walter
Molecular Biology of the Cell
Fifth Edition
Chapter 7
Control of Gene Expression
Copyright © Garland Science 2008
Summary
Endocrine system
adapted from http://www.clinica-verde.com/
A neuron and a lymphocyte share the same genome
Therefore, gene expression is affected and
regulated by a variety of external signals
Figure 7-1 Molecular Biology of the Cell (© Garland Science 2008)
‘Biologists originally suspected that genes
might be selectively lost when a cell
becomes specialized’.
‘All the cells contain the same genome.
Cell differentiation is achieved by changes
in gene expression’.
An Overview of Gene
Expression
Differentiated cells contain all the genetic instructions
necessary to direct the formation of a complete organism
• If DNA were altered irreversibly during development, the
chromosomes of a differentiated cell would be incapable of
guiding the development of the whole organism.
The different cell types of a multicellular
organism contain the same DNA
Figure 7-2a Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-2b Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-2c Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-3 Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-4 Molecular Biology of the Cell (© Garland Science 2008)
“Differentiated cells contain all the genetic
instructions necessary to direct the formation of
a complete organism”.
What makes cells express genes quite different
from each other?
A cell can change the expression of its
genes in response to external signals
Different cell types produce different sets
of proteins
• A typical differentiated human cell expresses perhaps
5,000-15,000 genes from a repertoire of about
25,000.
Gene expression can be regulated at many of the steps in
the pathway from DNA to RNA to protein
• Extracellular cues (starvation or intense exercise)
• Glucocorticoid hormones (liver cell)
• Increase the production of glucose from amino acids and other small
molecules.
• Induce enzymes such as tyrosine aminotransferase (which helps
to convert tyrosine to glucose).
• Glucocorticoid hormones (fat cells)
• Reduce production of tyrosine aminotransferse.
•
Some other cells
do not respond to glucocorticoids at all.
Figure 7-5 Molecular Biology of the Cell (© Garland Science 2008)
How Transcriptional Switches Work
What makes cells express genes
quite different from each other?
Transcription is controlled by proteins
binding to regulatory DNA sequences
REVIEW
Polymerase II also requires activator, mediator,
and chromatin modifying proteins
• Regulatory DNA sequences
•
In addition to the promoter, nearly
DNA sequences.
all genes have the regulatory
the gene on or off.
• Short (10 nt) : Simple gene switches.
• Very long (10 kbp) : molecular microprocessors.
• Recognized by gene regulatory proteins.
• Combination of a DNA sequence and its associated regulatory protein
•
Used to switch
molecules
→ Acts
as the switch to control transcription.
Gene regulatory protein (Transcription Factor)
• Several thousand proteins in human.
• Bind to the major groove of a DNA helix.
• Contain a variety of DNA-binding motifs.
1. Homeodomain : 3 linked α helices.
2. Zinc finger motif : an α helix and a β sheet.
3. Leucine
zipper motif : two α helices.
Table 7-1 Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-6 Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-7 Molecular Biology of the Cell (© Garland Science 2008)
Gene Regulatory Proteins Contain
A Gene Regulatory Protein Binds to the
major groove of a DNA helix.
DNA-binding Motifs
Homeodomain
Homeodomain
Zinc finger motif
Leucine zipper motif
Figure 7-9 Molecular Biology of the Cell (© Garland Science 2008)
Homeodomain
•
•
A structure motif in many eucaryotic DNA-binding proteins.
Three linked α helices.
• Most of the contacts are made by helix 3.
• Helix 3 makes important contact with the major groove of DNA (ex.
The Asn contacts an adenine).
• A member of the helix-turn-helix family.
Zinc Finger Domain
• Built from an α helix and a β sheet by a molecule of zinc.
• Often found in clusters.
• α helix
contacts the DNA bases.
• Recognize DNA using three zinc fingers of the Cys-Cys-HisHis type.
One type of zinc finger protein
(Cys-Cys-His-His family)
Figure 7-14 Molecular Biology of the Cell (© Garland Science 2008)
DNA binding by a zinc finger protein
Figure 7-15 Molecular Biology of the Cell (© Garland Science 2008)
Leucine Zipper Domain
• Two α helices.
• Bind to DNA as dimers.
• Each motif makes many contacts with DNA.
Figure 7-27a Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-27b Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-28 Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-29a Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-29b Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-30 Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-31 Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-32 Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-32 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-32 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-33 Molecular Biology of the Cell (© Garland Science 2008)
REVIEW
Transcriptional switches allow cells to
respond to changes in the environment
(Procaryotic cell)
•
•
•
Bacterium E. coli : single circular DNA of 4.6 x 106 nt.
Regulate the expression : food sources.
Example: Tryptophan
Operon
A set of genes → a single mRNA.
• Operon:
Common in bacteria.
•
• 5 genes code for enzymes for tryptophan.
Figure 7-34 Molecular Biology of the Cell (© Garland Science 2008)
Tryptophan Repressor
•
•
Repressor inactive.
RNA polymerase transcribes Try
genes.
•
•
•
Repressor active.
Binds to the operator.
Blocks the binding of RNA polymerase.
Figure 7-35 Molecular Biology of the Cell (© Garland Science 2008)
*Constitutive gene (cf. inducible gene):
Figure 7-36 Molecular Biology of the Cell (© Garland Science 2008)
Repressors turn genes off, activators turn
them on
• Continuously transcribed at a low level without regulation of gene expression (ex. Trp
repressor).
• The bacterium can respond very rapidly to the rise in tryptophan concentration.
Activator
☞ Allosteric protein:
• Binding of Trp causes a subtle change in its
three-dimensional structure of repressors.
• Repressors can now bind to the operator DNA.
• Switch / on.
• Regulatory sequence.
• Initiate transcription.
• Controlled by the interaction of a metabolite or other
small molecule.
• Example: CAP has to bind cAMP before it can bind to
DNA.
Activator
Figure 7-37 Molecular Biology of the Cell (© Garland Science 2008)
An activator and a repressor control the lac operon
Figure 7-38 Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-39 Molecular Biology of the Cell (© Garland Science 2008)
• Lac operon encodes proteins to digest lactose.
• Lac operon is controlled by lac repressor + activator CAP.
Control of Gene Expression
(Eucaryotic cell)
• glu • CAP switches on genes.
• Utilize alternative sources of carbon (lactose).
Regulatory DNA sequences
Nucleosome structure
• lac • lac repressor bound and the operon is shut off.
•
Eucaryotic transcription regulator control
gene expression from a distance
“It was surprising to biologists when, in 1979, it was
discovered that these activator proteins could be bound
thousands of nucleotide pairs away from the promoter”
• Activator: Aid the assembly of the GTFs and RNApII.
• Enhancers: DNA sites to which the activators bound.
Models for “Action at a distance”
• Loop out : the enhancer + the promoter
• DNA acts as a tether.
• Mediators: serves to ‘link’ the gene regulatory proteins to
the RNApII and GTFs.
The gene control region of a typical eucaryotic gene
A eucaryotic gene control region consists of
a promoter plus regulatory DNA sequences
• Gene looping
• Spacer DNA
•
provides the flexibility needed for efficient DNA looping.
•
Allows the genes of an organism to be turned on of off
individually.
•
Of the roughly 25,000 human genes, an estimated 8%
(~2,000 genes) encode gene regulatory proteins.
• Gene regulatory sequence
Figure 7-44 Molecular Biology of the Cell (© Garland Science 2008)
• Enhancers
•
•
The DNA sites to which eucaryotic gene activator proteins
bind.
•
Their presence “enhanced” the rate of transcription initiation.
The simplest gene activator proteins
distinct domains (DNA binding domain + activation
• Two
domain).
• A chimeric protein
Figure 7-45 Molecular Biology of the Cell (© Garland Science 2008)
Eucaryotic gene activator proteins promote the
assembly of RNA polymerase and the general
transcription factors at the startpoint of transcription
Figure 7-45a Molecular Biology of the Cell (© Garland Science 2008)
Packing of promoter DNA into nucleosomes
affects initiation of transcription
“Nucleosomes can inhibit the initiation of transcription
probably because they physically block the assembly of
the general transcription factors, RNA polymerase on
the promoter”
In eucaryotic cells, activator and repressor exploit
chromatin structure to help turn genes on and off.
Figure 7-45b Molecular Biology of the Cell (© Garland Science 2008)
1. Chromatin remodeling complex
• Change the structure of nucleosomes.
• ATP hydrolysis
• Accessibility ↑
2. Histone modifying proteins
• Histone acetyltransferase (HAT) (Gene activator)
Acetylation to K of histone; Accessibility ↑
Assembly of the GTFs and RNA pol II ↑ (Acetyl groups themselves are
recognized by proteins that promote transcription).
• Histone deacetylase (HDAC) (Gene repressor)
Deacetylation; Accessibility ↓
• HDAC inhibitors specifically inhibit the enzyme, histone
deacetylase in eucaryotic cells including most types of cancers.
• Today, HDAC inhibitors are being evaluated as anticancer
drugs with novel mechanism.
• What would be the proposed mechanisms by which
HDAC inhibitors suppress the growth of cancer cells including
‘Apoptosis’?
Figure 7-46 Molecular Biology of the Cell (© Garland Science 2008)
In depth study
•
Eucaryotic gene activator proteins also
modify local chromatin structure
•
General idea
•
The GTFs, mediator, and RNA polymerase seem unable on
their own to assemble on a promoter that is packed in
standard nucleosomes.
•
Such packing may have evolved to prevent “leaky”
transcription.
Four of the most important ways of locally
altering chromatin
• Nucleosome remodeling
• Histone modificaiton
• Nucleosome removal
• Nucleosome replacement
• Writing and reading the histone code during transcription
Gene activator proteins work
synergistically
•
Gene activator proteins often exhibit transcriptional
synergy, where several activator proteins working
together produce a transcription rate that is much
higher than that of the sum of the activators working
alone.
Figure 7-47 Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-47 (part 1 of 4) Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-47 (part 2 of 4) Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-47 (part 3 of 4) Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-47 (part 4 of 4) Molecular Biology of the Cell (© Garland Science 2008)
•
Figure 7-48 Molecular Biology of the Cell (© Garland Science 2008)
An order of events leading to transcription initiation
of a specific gene.
•
Does histone modification always precede
chromatin remodeling?
•
Does mediator enter before or after RNA
polymerase?
•
Different for different genes.
Eucaryotic gene repressor proteins can
inhibit transcription in various ways
• Competitive DNA binding
• Masking the activation surface
• Direct interaction with the general transcription factors
•
•
•
Recruitment of chromatin
deacetylase
Recruitment of histone
methyl transferase
Figure 7-49 Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-50a Molecular Biology of the Cell (© Garland Science 2008)
remodeling complexes
Recruitment of histone
Figure 7-50b Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-50c Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-50d Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-50e Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-50f Molecular Biology of the Cell (© Garland Science 2008)
The Molecular Mechanism That Create
Specialised Cell Type
choice of fate
is generally maintained through many subsequent cell generations.
•
Once a cell becomes committed to differentiate into a specific cell type, the
•
Changes in gene expression involved in the choice must be remembered.
• ‘cell memory’
• cf. Transient regulation
Eucaryotic genes are regulated by combinations of proteins
• Combinatorial control:
: Groups of proteins work together for the
expression of a single gene.
• Now beginning
: How to determine the final level of expression
for a gene are only to be understood...
of gene expression (ex, tryptophan repressor).
The expression of different genes can be
coordinated by a single protein
Glucocorticoids (GCs)
- GC is a class of steroid hormones that bind to the glucocorticoid receptor.
Switch on/off
bac cells
euk cells
Individually
√
√
combinatorially
operon
?
- GC is present in almost every vertebrate animal cell.
- Glucocorticoid (glucose + cortex + steroid) : its metabolism regulation of
glucose, its synthesis in the adrenal cortex, and its steroidal structure.
- GC turns immune activity (inflammation) down.
- GCs are therefore used in medicine to treat diseases caused by an
overactive immune system, such as allergies, asthma, autoimmune
diseases, and sepsis.
- Cortisol is the most important human glucocorticoid.
•
•
In particular, a eucaryotic cell uses
to control each of its genes.
How can it
a committee of regulatory proteins
- Various synthetic glucocorticoids to treat glucocorticoid deficiency or to
suppress the immune system.
rapidly and decisively switch whole groups of genes on or off?
http://en.wikipedia.org/wiki/Glucocorticoid
• A single gene regulatory protein
• ‘decisive’
•Completing
•Switching
the combination to activate or repress.
any particular gene on or off.
• Complete the combination for
ex) glucocorticoid receptor protein
1. at low level: bound activators are not sufficient on their own to activate tx.
2. at high level: The glucocorticoid receptor completes the combination of gene
regulatory proteins required for efficient initiation of tx.
several different genes.
Combinational control can create different cell types
A single gene regulatory protein vs differentiation
A mammalian skeletal muscle cell
extremely large cell.
fusion of many myoblast.
Genes are all switched on coordinately as the myoblasts begin to
•
An
•
Formed by the
•
fuse.
• Actin, myosin, receptor proteins, ion channel proteins.
http://snap.yescall.com/admin/com/d_9675/462973_b.jpg
myoD
• Key gene regulatory protein.
Coordinates the gene expression.
muscle cell differentiation.
regulatory regions.
• Activates the tx of the genes that code for the muscle-specific proteins.
• Converts nonmuscle cells to myoblasts (fibroblast to myoblast)
•
• Crucial for
• Binds to their
• Activates the changes in gene expression typical of differentiating muscle cells.
→ The fibroblast have already accumulated all of the other
necessary gene regulatory proteins required for the combinatorial control
of the muscle-specific genes, and that addition of MyoD completes the unique
combination that directs the cells to become muscle.
Stable patterns of gene expression can be
transmitted to daughter cells
1. ‘Positive feedback loop’
• A key gene regulatory protein activates transcription
of its own gene.
• MyoD
• Once a cell has become differentiated...
• Generally remain differentiated.
• All its progeny cells will be of that same cell type.
•Remembered.
• How do cells ensure that daughter cells “remember”
what kind of cells they are supposed to be?
2. Condensed chromatin structure
• States of chromatin structure can be inherited.
transferred
• A cluster of chromatin proteins
bound to the DNA are
(epigenetic regulation).
• X chromosome inactivation.
3. DNA methylation
• DNA methylation on Cytosine bases (Covalent modification).
3. DNA methylation
• DNA methylation on Cytosine bases (Covalent modification).
• Generally turns off genes by attracting proteins that block gene expression.
• Passed on to progeny cells by an enzyme that copies the pattern
to the daughter DNA strand immediately after replication.
*Epigenetic inheritance
: transmits information from parent to daughter cell without
altering the actual nucleotide sequence of DNA (such as positive feedback, certain forms of condensed
chromatin, and DNA methylation)
The formation of an entire organ can be
triggered by a single gene regulatory protein
• A single gene regulatory protein
(Ey in flies, Pax-6 in
vertebrates)
• Trigger the formation of not just a single cell type but a whole organ – the eye (composed
of different types of cells all properly organized in three dimensional space).
•Turns on a cascade of gene regulatory proteins
.
• Forms an organized group of many different types of cells.
• How does the Ey protein coordinate the specification of
each cell in the eye?
Post-transcriptional Controls
• An actively studied topic in developmental biology.
•Ey directly controls
the expression of many genes by binding to DNA sequences in their
regulatory regions.
• Some of the genes controlled by Ey encode additional transcription regulators
that control the expression of other genes.
• Some of these regulators act back on Ey itself to create a positive feedback loop that
ensures the continued production of the Ey protein.
• Therefore, the action of just one transcription regulator can produce
a cascade of regulators whose combined actions lead to the formation of an organized group
of many different types of cells.
• Operate after RNA polymerase has bound to a gene’s
promoter and started to synthesize RNA.
Alternative splicing.
More examples
•
•
Riboswitch; UTR of mRNA;
miRNA; siRNA
review
Riboswitches provide an economical solution
to gene regulation
• Riboswitches
• Complex folded RNA domains.
• Serve as receptors for specific metabolites.
• Found in non-coding portion
•Control gene expression
of various mRNAs.
by allosteric structural changes (metabolite binding).
• A double-stranded structure that forces the polymerase to terminate
transcription.
Bypass the need for regulatory proteins altogether (Economical examples of gene
•
•Robust genetic elements
control).
The untranslated region (UTR) of mRNAs can
control their translation
• Gene expression can be controlled by regulating
translation initiation (Bacteria)
• Sequence-specific RNA-binding proteins.
• Thermosensor RNA sequences.
• Riboswitches.
• Antisense RNA.
in many organisms.
Sequence-specific RNA-binding proteins
Thermosensor RNA sequences
Riboswitches
microRNA (miRNA)
Small regulatory RNAs control the expression
of thousands of animal and plant genes
Antisense RNA
• microRNA (miRNA)
•Noncoding RNAs
transcribed by Pol II.
• More than 400 different miRNAs in human.
•Regulate at least one-third
of all protein-coding genes.
• Base-paring with specific mRNA s and controlling their stability and
translation.
• Form an RNA-induced silencing complex (RISC).
Target mRNA is destroyed immediately by a nuclease present within the RISC.
•
• What features of miRNA make them especially useful
regulators of gene expression?
• First, a single miRNA can regulate a whole set of different mRNA so
long as the mRNAs carry a common sequence (5’ and 3’ UTRs; some individual miRNAs control
hundreds of different mRNAs in human).
• Second, a gene that encodes an miRNA occupies relatively little space in the
genome compared with one that encodes a transcription regulator (Small size).
RNA interference destroys double-stranded
foreign RNAs
small interfering RNAs (siRNAs)
• RNA interference (RNAi)
• A cell defense mechanism.
• Orchestrate the destruction of ‘foreign’ double-stranded RNA (From
viruses or transposable genetic elements; Targeted RNA degradation mechanism).
• RNAi response resembles human immune system.
• Action mechanism by which siRNA destroy foreign RNAs.
•Foreign RNA attracts Dicer
•Dicer cleaves the double-stranded RNA
(a protein complex with nuclease).
into short fragments (about 23 nt);
small interfering RNAs (siRNAs).
• siRNAs (short, double-stranded RNAs) are then incorporated into RISCs.
• RISC discard one strand and uses the remaining single-stranded RNA to locate a
complementary foreign RNA molecule.
• This targeted RNA molecule is then rapidly degraded.
•RNAi
powerful experimental tool to inactivate almost any
has become
gene in cells (Chapter 10).
• RNAi as a powerful new approach for treating human disease
through the regulation of gene expression (siRNA for medical promise).
• RNAi expands our understanding of the types of regulatory networks to
specify the development of complex organisms, including ourselves.
Scientists can use RNA interference to turn
off genes