Chapter 17
Gene Regulation
in Eukaryotes
Similarity of regulation between
eukaryotes and prokaryote
1,Principles are the same,signals,
activators and repressors,recruitment
and allostery,cooperative binding
2,Expression of a gene can be regulated
at the similar steps,and the initiation
of transcription is the most pervasively
regulated step.
Difference in regulation between
eukaryotes and prokaryote
1,Pre-mRNA splicing adds an important
step for regulation.
2,The eukaryotic transcriptional machinery
is more elaborate than its bacterial
counterpart.
3,Nucleosomes and their modifiers
influence access to genes.
4,Many eukaryotic genes have more
regulatory binding sites and are
controlled by more regulatory proteins
than are bacterial genes.
A lot more regulator bindings sites in
multicellular organisms reflects the
more extensive signal integration
Fig,17-1
Bacteria
Yeast
Human
Enhancer,a given site binds regulator
responsible for activating the gene.
Alternative enhancer binds different
groups of regulators and control
expression of the same gene at
different times and places in responsible
to different signals.
Activation at a distance is much more
common in eukaryotes,Insulators (绝缘体 )
or boundary elements are regulatory
sequences to ensure a linked promoter
not responding to the activator binding,
Topic 1
Conserved Mechanisms of
Transcriptional Regulation
from Yeast
to Mammals
The basic features of gene regulation are
the same in all eukaryotes,because of
the similarity in their transcription and
nucleosome structure.
Yeast is the most amenable to both
genetic and biochemical dissection,and
produces much of knowledge of the action
of the eukaryotic repressor and activator,
The typical eukaryotic activators works in
a manner similar to the simplest bacterial
case.
Repressors work in a variety of ways
1-1 Eukaryotic activators have
separate DNA binding and
activating functions,which are very
often on separate domains of the protein.
Fig,17-2 Gal4 bound to its site on DNA
Fig,17-3 The regulatory sequences of the
Yeast GAL1 gene.
1.Gal4 is the most studied eukaryotic activator
2.Gal4 activates transcription of the galactose
genes in the yeast S,cerevisae.
3.Gal4 binds to four sites upstream of GAL1,
and activates transcription 1,000-fold in the
presence of galactose
The separate DNA binding and activating
domains of Gal4 were revealed in two
complementary experiments
1,Expression of the N-terminal region
(DNA-binding domain) of the activator
produces a protein bound to the DNA
normally but did not activate
transcription.
2,Fusion of the C-terminal region
(activation domain) of the activator to
the DNA binding domain of a bacterial
repressor,LexA activates the
transcription of the reporter gene,
Domain swap experiment
Domain swap
experiment
Moving domains among
proteins,proving that
domains can be
dissected into
separate parts of the
proteins.
Many similar
experiments shows
that DNA binding
domains and
activating regions
are separable.
Box 1 The two hybrid Assay is used to
identify proteins interacting with each other.
1-2 Eukaryotic regulators use a
range of DNA binding domains,
but DNA recognition involves
the same principles same
found in bacteria
Homeodomain proteins
Zinc containing DNA-binding domain,
zinc finger and zinc cluster
Leucine zipper motif
Helix-Loop-Helix proteins, basic zipper
and HLH proteins
Bactrial regulatory proteins
? Most use the helix-turn-helix motif to
bind DNA target
? Most bind as dimers to DNA sequence,
each monomer inserts an a helix into the
major groove.
Eukaryotic regulatory proteins
1,Recognize the DNA using the similar
principles,with some variations in detail.
2,Some form heterodimers to recognize
DNA,extending the range of DNA-
binding specificity,
Homeodomain proteins,The homeodomain
is a class of helix-turn-helix DNA-binding
domain and recognizes DNA in essentially
the same way as those bacterial proteins
Figure 17-5
Zinc containing DNA-binding domains
finger domain,Zinc finger proteins
(TFIIIA) and Zinc cluster domain (Gal4)
Figure 17-6
Leucine Zipper Motif,The Motif
combines dimerization and DNA-binding
surfaces within a single structural unit.
Figure 17-7
Dimerization is mediated by
hydrophobic interactions between
the appropriately-spaced leucine
to form a coiled coil structure
Helix-Loop-Helix motif:
Figure 17-8
Because the region of the a-helix
that binds DNA contains baisc
amino acids residues,Leucine
zipper and HLH proteins are often
called basic zipper and basic HLH
proteins.
Both of these proteins use
hydrophobic amino acid residues
for dimerization.
1-3 Activating regions are
not well-defined
structures
The activating regions are grouped
on the basis of amino acids content
? Acidic activation domains
? Glutamine-rich domains
? Proline-rich domains
Ⅱ Recruitment of
Protein Complexes
to Genes
by
Eukaryotic Activation
Eukaryotic activators also work by
recruiting as in bacteria,but recruit
polymerase indirectly in two ways,
2-1 Interacting with parts of the
transcription machinery.
2-2 Recruiting nucleosome
modifiers that alter chromatin in
the vicinity of a gene.
The eukaryotic transcriptional machinery
contains polymerase and numerous proteins
being organized to several complexes,such as
the Mediator and the TFⅡ D complex,
Activators interact with one or more of these
complexes and recruit them to the gene.
Figure 17-9
Box 2 Chromatin Immuno-precipitation (ChIP)
to visualize the recruitment (Where a given
protein is bound in the genome of a living cell.)
Activator Bypass Experiment-Activation of
transcription through direct tethering of
mediator to DNA,(很有应用价值 )
Directly fuse the
bacterial DNA-
binding protein
LexA protein to
the mediator
complex Gal11 to
activate GAL1
expression,
Figure 17-10
At most genes,the transcription
machinery is not prebound,and
appear at the promoter only upon
activation,Thus,no allosteric
activation of the prebound
polymerase has been evident in
eukaryotic regulation
2-2 Activators also recruit
modifiers that help the
transcription machinery bind at
the promoter
1,Modifiers direct recruitment of the
transcriptional machinery
2,Modifiers help activate a gene
inaccessibly packed within chromatin
Two types of Nucleosome modifiers,
Those add chemical groups to the tails
of histones,such as histone acetyl
transferases (HATs)
Those remodel the nucleosomes,such
as the ATP-dependent activity of
SWI/SNF
How do these modification help activate
a gene?
Two basic models for how these
modification help activate a gene,
Remodeling and certain modification
can uncover DNA-binding sites that
would otherwise remain inaccessible
within the nucleosome.
By adding acetyl groups,it creates
specific binding sites on
nucleosomes for proteins bearing
so-called bromodomains.
Figure 7-39 Effect of histone tail modification
Fig 17-11 Local alterations in chromatin
directed by activators
Many enkaryotic activators－ particularly
in higher eukaryotes－ work from a distance.
Why?
1,Some proteins help,for example Chip
protein in Drosophila,
2,The compacted chromosome structure help,
DNA is wrapped in nucleosomes in
eukaryotes.So sites separated by many base
pairs may not be as far apart in the cell as
thought.
2-2 Action at a distance,loops
and insulators
Specific elements called
insulators control the actions of
activators,preventing the
activating the non-specific genes
Insulators
block
activation
by
enhancers
Figure 17-12
Transcriptional Silencing
Silencing is a specializes form of
repression that can spread along
chromatin,switching off multiple genes
without the need for each to bear
binding sites for specific repressor.
Insulator elements can block this
spreading,so insulators protect genes
from both indiscriminate activation and
repression.So a gene inserted at random
into the mammalian genome is often
“silenced”.
2-4 Appropriate regulation of some
groups of genes requires locus control
region (LCR).
Figure 17-13
A group of regulatory elements collectively
called the locus control region (LCR),is
found 30-50 kb upstream of the cluster of
globin genes,It’s made up of multiple-
sequence elements, something like
enhancers,insulators or promoters.
It binds regulatory proteins that cause the
chromatin structure to,open up”,allowing
access to the array of regulators.
Another group of mouse genes whose
expression is regulated in a temporarily
and spatially ordered sequence are
called HoxD genes,They are controlled
by an element called the GCR (global
control region) in a manner very like
that of LCR.
Ⅲ Signal Integration
and
Combinatorial
Control
3-1 Activators work together
synergistically (协同的 ) to
integrate signals
In eukaryotic cells,numerous signals are often
required to switch a gene on,So at many
genes multiple activators must work together.
They do these by working synergistically,two
activators working together is greater than
the sum of each of them working alone.
Three strategies of synergy,
Two activators recruit a single complex
Activators help each other binding
cooperativity
One activator recruit something that helps
the second activator bind
a.“Classical”
cooperative
binding
b,Both
proteins
interacting
with a third
protein
c,A protein recruits
a remodeller to
reveal a binding site
for another protein
d,Binding a
protein unwinds
the DNA from
nucleosome a
little,revealing
the binding site
for another
proteinFigure 17-14
3-2 Signal integration,the HO
gene is controlled by two
regulators; one recruits
nucleosome modifiers and the
other recruits mediator
The HO gene is involved in the budding of yeast.
It has two activators, SWI5 and SBF.
alter the
nucleosome
Figure 17-15
3-3 Signal integration,
Cooperative binding of
activators at the human b-
interferon gene.
The human β-interferon gene is activated in cells
upon viral infection,Infection triggers three
activators,
NFκB,IRF,
and Jun/ATF,
They bind
cooperatively
to sites within
an enhancer,
form a
structure
called
enhanceosome.
Figure 17-16
3-4 Combinatory control lies at
the hear of the complexity and
diversity of eukaryotes
There is extensive combinatorial control in
eukaryotes.
In complex multicellular organisms,combinatorial
control involves many more regulators and genes
than shown above,and repressors as well as
activators can be involved,
Four
signals
Three
signals
Figure 17-17
3-5 Combinatory control of the
mating-type genes from S,
cerevisiae (啤酒酵母 )
The yeast S.cerevisiae exists in three
forms,two haploid cells of different
mating types－ a and a － and the diploid
formed when an a and an a cell mate
and fuse.
Cells of the two mating types differ
because they express different sets of
genes, a specific genes and a specific
genes.
a cell make the regulatory protein a1,
a cell make the protein a1 and a2.
A fourth regulator protein Mcm1 is
also involved in regulatory the mating-
type specific genes and is present in
both cell types.
How do these regulators work together
to keep a cell in its own type?
Control of cell-type specific genes in yeast
Figure 17-18
Ⅳ
Transcriptional
Repressors
In eukaryotes,repressors don’t work
by binding to sites that overlap the
promoter and thus block binding of
polymerase,but most common work by
recruiting nucleosome modifiers.
For example,histone deacetylases
repress transcription by removing
actetyl groups from the tails of histone.
Ways in
which
eukaryotic
repressor
Work
a and b
Figure 17-19
Ways in
which
eukaryotic
repressor
Work
c and d
Silencing
In the presence of glucose,Mig1 binds
a site between the USAG and the GAL1
promoter,By recruiting the Tup1
repressing complex,Mig1 represses
expression of GAL1,
A specific example,Repression of
the GAL1 gene in yeast
Ⅴ Signal Transduction
and
the Control of
Transcriptional Regulators
5-1 Signals are often
communicated to
transcriptional regulators
through signal transduction
pathway
Signals refers to initiating ligand (can
be sugar or protein or others),or just
refers to,information”.
There are various ways that signals
are detected by a cell and
communicated to a gene,But they are
often communicated to transcriptional
regulators through signal transduction
Pathway,in which the initiating ligand is
detected by a specific cell surface
receptor.
In a signal transduction pathway:
initiating ligand binds to an
extracellular domain of a specific cell
surface receptor this binding
bring an allosteric change in the
intracellular domain of receptor
the signal is relayed to the relevant
transcriptional regulator often
through a cascade of kinases.
5-2 Signals control the
activities of eukaryotic
transcriptional regulators in a
variety of ways
a,The STAT pathway
b,The MAP kinase pathway
Once a signal has been communicated,
directly or indirectly,to a transcriptional
regulator,how does it control the
activity of that regulator?
In eukaryotes,transcriptional regulators
are not typically controlled at the level
of DNA binding,They are usually
controlled in one of two basic ways,
Unmasking an activating region
Transport in or out of the nucleus
Activator Gal4 is regulated by masking protein Gal80
The signalling ligand causes activators (or
repressors) to move to the nucleus where
they act from cytoplasm.
5-3 Activators and repressors
sometimes come in pieces.
For example,the DNA binding domain
and activating region can be on
different polypeptides,same of an
activator
In addition,the nature of the protein
complexes forming on DNA
determines whether the DNA-binding
protein activates or represses nearby
genes,For example,the
glucocorticoid receptor (GR).
Ⅵ Gene,Silencing”
by
Modification of
Histones and DNA
Gene,silencing” is a position effect－ a
gene is silenced because of where it
is located,not in response to a
specific environmental signal.
The most common form of silencing is
associated with a dense form of
chromatin called heterochromatin,It
is frequently associated with
particular regions of the chromosome,
notably the telomeres,and the
centromeres.
6-1 Silencing in yeast is
mediated by deacetylation ane
methylation of the histones
The telomeres,the silent mating-type locus,
and the rDNA genes are all,silent” regions in
S.cerevisiae.
Three genes encoding regulators of silencing,
SIR2,3,and 4 have been found (SIR stand
for silent information regulator).
Silencing at the yeast telomere
6-1 Histone modification and
the histone code hypothesis
A histone code exists?
According to this idea,different
patterns of modification on histone tails
can be,read” to mean different things,
The,meaning” would be the result of
the direct effects of these
modifications on chromatin density and
form,
But in addition,the particular pattern
of modifications at any given location
would recruit specific proteins.
Transcription can also be silenced by
methylation of DNA by enzymes
called DNA methylases.
This kind of silencing is not found in
yeast but is common in mammalian
cells.
Methylation of DNA sequence can
inhibit binding of proteins,including
the transcriptional machinery,and
thereby block gene expression.
Switching a gene off,
A mammalian gene marked by methylation
of nearby DNA sequence
recognized by DNA-binding proteins
recruit histone decetylases and histone
methylases
modify nearby chromatin
This gene is completely off.
Switching a gene off
Figure 17-24
DNA methylation lies at the heart of a
phenomenon called imprinting,
Two examples,Human H19 and Igf2 genes.
Here an enhancer and an insulator are critical.
Patterns of gene expression must
sometimes be inherited,These
may remain for many cell generations,
even if the signal that induced them is
present only fleetingly,
This inheritance of gene expression
patterns is called epigenetic regulation.
Maintenance of a phage λlysogen,can
be described as an example.
λlysogens and the epigentic switchBox 3
Lysogenic gene expression is established in an
infected cell in response to poor growth
conditions,Then the lysogenic state will remain
through cell
division in
both cells,
This is
resulted from
a two-step
strategy for
repressor
synthesis.
How?
Nucleosome and DNA modifications
can provide the basis for epigenetic
inheritance.
DNA methylation is even more reliably
inherited,but far more efficiently is
the so-called maintenance methylases
modify hemimethylated DNA－ the very
substrate provided by replication of
fully methylated DNA.
Patterns of DNA methylation can be
maintained through cell division
Ⅶ Eukaryotic
Gene Regulation
at Steps
after
Transcription Initiation
In eukaryotic cells,some regulational
proteins aim at elongation.
At some genes there are sequence
downstream of the promoter that
cause pausing or stalling of the
polymerase soon after initiation.
At those genes,the presence or
absence of certain elongation
factors greatly influences the level
at which the gene is expressed.
Two examples, HSP70 gene and HIV
HIV genome
?
Many individual eukaryotic genes have exons
interrupted by introns,So when the whole gene
is transcribed,mRNA need to be spliced.
In some cases a given precursor mRNA
can be spliced in alternative ways to
produce different mRNAs that encode
different protein products.
The regulation of alternative splicing
works in a manner reminisencent, the
splicing machinery binds to splice sites
and carries out the splicing reaction.
The sex of a fly is determined by the
ratio of X chromosomes to autosomes,
This ratio is initially measured at the
level of transcription using two
activators SisA and SisB,The genes
encoding these regulators are both on
the X chromosome.
Sxl, Sex-lethal
Dpn,Deadpan
Early transcriptional regulation of Sxl in
male and female flies
A cascade
of
alternative
splicing
events
determines
the sex
of a fly
Gcn4 is a yeast transcriptional activator
that regulates the expression of genes
encoding enzymes that direct amino acid
biosynthesis,
The mRNA encoding the Gcn4 protein
contains four small open reading frames
(called uORFs) upstream of the coding
sequence for Gcn4.
Although it is a activator,Gcn4 is itself
regulated at the level of translation,
In the presence of low levels of amino
acids,the Gcn4 mRNA is translated
(and so the biosynthetic are
expressed),
In the presence of high levels,it is
not translated.
How is this regulation achieved?
high levels
of amino
acids,
the Gcn4
mRNA
is not
translated
low levels
of amino
acids,
the Gcn4
mRNA is
translated
Ⅷ RNAs
in
Gene Regulation
8-1 Double-standed RNA
inhibits expression of genes
homologous to that RNA
8-2 Short interfering RNA
(siRNAs) are produced from
dsRNA and direct machinery
that switch off genes in various
way
8-3 MicroRNA control the
expression of some genes
during development.
Short RNAs can direct repression of
genes with homology to those short
RNAs,
This repression,called RNA
interference (RNAi),can manifest as
translational inhibition of the mRNA,
destruction of the mRNA or
transcriptional silencing of the
promoter that directs expression of
that mRNA.
The discovery that simply introducing
double-strand RNA (dsRNA) into a cell
can repress genes containing sequence
identical to (or very similar to) that
dsRNA was remarkable in 1998 when
it was reported.
A similar effect is seen in many other
organisms in both animals and plants.
How dsRNA can switch off expression
of a gene?
Dicer is an RNAseⅢ -like enzyme that
recognizes and digests long dsRNA,The
products are short double-stranded fragments.
These short RNAs (or short interfering RNAs,
siRNAs) inhibit expression of a homologous gene
in three ways,
Trigger destruction of its mRNA
Inhibit translation of its mRNA
Induce chromatin modifications within the
promoter that silence the gene
That machinery includes a complex called RISC
(RNA-induced silencing complex).
RNA
silencing
RNAi silencing is extreme efficiency.
Very small amounts of dsRNA are
enough to induce complete shutdown
of target genes.
Why the effect is so strong?
It might involve an RNA-dependent
RNA polymerase which is required in
many cases of RNAi.
There is another class of naturally
occurring RNAs,called microRNAs
(miRNAs),that direct repression of
genes in plants and worms.
Often these miRNAs are expressed in
developmentally regulated patterns.
The mechanism of RNAi may have
evolved originally to protect cells from
any infectious,or otherwise disruptive,
element that employs a dsRNA
intermediate in its replicative cycle.
Now RNAi has been adapted for use as
a powerful experimental technique
allowing specific genes to be switched
off in any of many organisms.
Summary,
There are several complexities in the
organization and transcription of
eukaryotic genes not found in bacteria,
Nucleosomes and their modification
Many regulators and larger distances
The elaborate transcriptional machinery
Pay attention to these differences,and tell
them in details by yourself.
Do you know these conceptions?
Promoter
Regulator binding site
Regulatory sequence
Enhancer
Insulator
Reporter gene
Gene silencing
Critical Thinking Exercises
1,Compare the mechanisms used by zinc-binding,leucine zipper,
and HLH motifs to bind DNA,What is the role of zinc in the
zinc-binding domain?
2,Describe the physical characteristics of a typical transcriptional
activation region,How are these characteristics thought to
reflect the mechanism of activation? If a novel transcription
factor is identified,and you would like to locate the domain
within the protein that is responsible for activation,how would
you do this?
3,You transform a population of cells with a transgene,and
isolate a cell line that has integrated the gene into its genome,
but in which the gene is not expressed,Speculate as to what
may be preventing the gene from being expressed,Describe
two ways to test this possibility,
4,To determine how an activator contributes to the formation
of the holoenzyme at your favorite promoter,design an
experimental strategy to tell you which components are
directly recruited by the activator,and which of these
activator-mediated recruitment steps are required for
transcriptional activation,How could you test whether the
role of the activator stops with the holoenzyme assembly,
or if it also induces allosteric changes within the DNA or the
holoenzyme?
5,You isolate a mutant strain of mice that grow at an
unusually fast rate,perform a blood test on the mice,and
find that they have elevated levels of insulin-like growth
factor,The phenotype is the result of a mutation in a region
of the genome containing a gene encoding a DNA
methylase,What kind of mutation is causing the rapid
growth of these mice?
Gene Regulation
in Eukaryotes
Similarity of regulation between
eukaryotes and prokaryote
1,Principles are the same,signals,
activators and repressors,recruitment
and allostery,cooperative binding
2,Expression of a gene can be regulated
at the similar steps,and the initiation
of transcription is the most pervasively
regulated step.
Difference in regulation between
eukaryotes and prokaryote
1,Pre-mRNA splicing adds an important
step for regulation.
2,The eukaryotic transcriptional machinery
is more elaborate than its bacterial
counterpart.
3,Nucleosomes and their modifiers
influence access to genes.
4,Many eukaryotic genes have more
regulatory binding sites and are
controlled by more regulatory proteins
than are bacterial genes.
A lot more regulator bindings sites in
multicellular organisms reflects the
more extensive signal integration
Fig,17-1
Bacteria
Yeast
Human
Enhancer,a given site binds regulator
responsible for activating the gene.
Alternative enhancer binds different
groups of regulators and control
expression of the same gene at
different times and places in responsible
to different signals.
Activation at a distance is much more
common in eukaryotes,Insulators (绝缘体 )
or boundary elements are regulatory
sequences to ensure a linked promoter
not responding to the activator binding,
Topic 1
Conserved Mechanisms of
Transcriptional Regulation
from Yeast
to Mammals
The basic features of gene regulation are
the same in all eukaryotes,because of
the similarity in their transcription and
nucleosome structure.
Yeast is the most amenable to both
genetic and biochemical dissection,and
produces much of knowledge of the action
of the eukaryotic repressor and activator,
The typical eukaryotic activators works in
a manner similar to the simplest bacterial
case.
Repressors work in a variety of ways
1-1 Eukaryotic activators have
separate DNA binding and
activating functions,which are very
often on separate domains of the protein.
Fig,17-2 Gal4 bound to its site on DNA
Fig,17-3 The regulatory sequences of the
Yeast GAL1 gene.
1.Gal4 is the most studied eukaryotic activator
2.Gal4 activates transcription of the galactose
genes in the yeast S,cerevisae.
3.Gal4 binds to four sites upstream of GAL1,
and activates transcription 1,000-fold in the
presence of galactose
The separate DNA binding and activating
domains of Gal4 were revealed in two
complementary experiments
1,Expression of the N-terminal region
(DNA-binding domain) of the activator
produces a protein bound to the DNA
normally but did not activate
transcription.
2,Fusion of the C-terminal region
(activation domain) of the activator to
the DNA binding domain of a bacterial
repressor,LexA activates the
transcription of the reporter gene,
Domain swap experiment
Domain swap
experiment
Moving domains among
proteins,proving that
domains can be
dissected into
separate parts of the
proteins.
Many similar
experiments shows
that DNA binding
domains and
activating regions
are separable.
Box 1 The two hybrid Assay is used to
identify proteins interacting with each other.
1-2 Eukaryotic regulators use a
range of DNA binding domains,
but DNA recognition involves
the same principles same
found in bacteria
Homeodomain proteins
Zinc containing DNA-binding domain,
zinc finger and zinc cluster
Leucine zipper motif
Helix-Loop-Helix proteins, basic zipper
and HLH proteins
Bactrial regulatory proteins
? Most use the helix-turn-helix motif to
bind DNA target
? Most bind as dimers to DNA sequence,
each monomer inserts an a helix into the
major groove.
Eukaryotic regulatory proteins
1,Recognize the DNA using the similar
principles,with some variations in detail.
2,Some form heterodimers to recognize
DNA,extending the range of DNA-
binding specificity,
Homeodomain proteins,The homeodomain
is a class of helix-turn-helix DNA-binding
domain and recognizes DNA in essentially
the same way as those bacterial proteins
Figure 17-5
Zinc containing DNA-binding domains
finger domain,Zinc finger proteins
(TFIIIA) and Zinc cluster domain (Gal4)
Figure 17-6
Leucine Zipper Motif,The Motif
combines dimerization and DNA-binding
surfaces within a single structural unit.
Figure 17-7
Dimerization is mediated by
hydrophobic interactions between
the appropriately-spaced leucine
to form a coiled coil structure
Helix-Loop-Helix motif:
Figure 17-8
Because the region of the a-helix
that binds DNA contains baisc
amino acids residues,Leucine
zipper and HLH proteins are often
called basic zipper and basic HLH
proteins.
Both of these proteins use
hydrophobic amino acid residues
for dimerization.
1-3 Activating regions are
not well-defined
structures
The activating regions are grouped
on the basis of amino acids content
? Acidic activation domains
? Glutamine-rich domains
? Proline-rich domains
Ⅱ Recruitment of
Protein Complexes
to Genes
by
Eukaryotic Activation
Eukaryotic activators also work by
recruiting as in bacteria,but recruit
polymerase indirectly in two ways,
2-1 Interacting with parts of the
transcription machinery.
2-2 Recruiting nucleosome
modifiers that alter chromatin in
the vicinity of a gene.
The eukaryotic transcriptional machinery
contains polymerase and numerous proteins
being organized to several complexes,such as
the Mediator and the TFⅡ D complex,
Activators interact with one or more of these
complexes and recruit them to the gene.
Figure 17-9
Box 2 Chromatin Immuno-precipitation (ChIP)
to visualize the recruitment (Where a given
protein is bound in the genome of a living cell.)
Activator Bypass Experiment-Activation of
transcription through direct tethering of
mediator to DNA,(很有应用价值 )
Directly fuse the
bacterial DNA-
binding protein
LexA protein to
the mediator
complex Gal11 to
activate GAL1
expression,
Figure 17-10
At most genes,the transcription
machinery is not prebound,and
appear at the promoter only upon
activation,Thus,no allosteric
activation of the prebound
polymerase has been evident in
eukaryotic regulation
2-2 Activators also recruit
modifiers that help the
transcription machinery bind at
the promoter
1,Modifiers direct recruitment of the
transcriptional machinery
2,Modifiers help activate a gene
inaccessibly packed within chromatin
Two types of Nucleosome modifiers,
Those add chemical groups to the tails
of histones,such as histone acetyl
transferases (HATs)
Those remodel the nucleosomes,such
as the ATP-dependent activity of
SWI/SNF
How do these modification help activate
a gene?
Two basic models for how these
modification help activate a gene,
Remodeling and certain modification
can uncover DNA-binding sites that
would otherwise remain inaccessible
within the nucleosome.
By adding acetyl groups,it creates
specific binding sites on
nucleosomes for proteins bearing
so-called bromodomains.
Figure 7-39 Effect of histone tail modification
Fig 17-11 Local alterations in chromatin
directed by activators
Many enkaryotic activators－ particularly
in higher eukaryotes－ work from a distance.
Why?
1,Some proteins help,for example Chip
protein in Drosophila,
2,The compacted chromosome structure help,
DNA is wrapped in nucleosomes in
eukaryotes.So sites separated by many base
pairs may not be as far apart in the cell as
thought.
2-2 Action at a distance,loops
and insulators
Specific elements called
insulators control the actions of
activators,preventing the
activating the non-specific genes
Insulators
block
activation
by
enhancers
Figure 17-12
Transcriptional Silencing
Silencing is a specializes form of
repression that can spread along
chromatin,switching off multiple genes
without the need for each to bear
binding sites for specific repressor.
Insulator elements can block this
spreading,so insulators protect genes
from both indiscriminate activation and
repression.So a gene inserted at random
into the mammalian genome is often
“silenced”.
2-4 Appropriate regulation of some
groups of genes requires locus control
region (LCR).
Figure 17-13
A group of regulatory elements collectively
called the locus control region (LCR),is
found 30-50 kb upstream of the cluster of
globin genes,It’s made up of multiple-
sequence elements, something like
enhancers,insulators or promoters.
It binds regulatory proteins that cause the
chromatin structure to,open up”,allowing
access to the array of regulators.
Another group of mouse genes whose
expression is regulated in a temporarily
and spatially ordered sequence are
called HoxD genes,They are controlled
by an element called the GCR (global
control region) in a manner very like
that of LCR.
Ⅲ Signal Integration
and
Combinatorial
Control
3-1 Activators work together
synergistically (协同的 ) to
integrate signals
In eukaryotic cells,numerous signals are often
required to switch a gene on,So at many
genes multiple activators must work together.
They do these by working synergistically,two
activators working together is greater than
the sum of each of them working alone.
Three strategies of synergy,
Two activators recruit a single complex
Activators help each other binding
cooperativity
One activator recruit something that helps
the second activator bind
a.“Classical”
cooperative
binding
b,Both
proteins
interacting
with a third
protein
c,A protein recruits
a remodeller to
reveal a binding site
for another protein
d,Binding a
protein unwinds
the DNA from
nucleosome a
little,revealing
the binding site
for another
proteinFigure 17-14
3-2 Signal integration,the HO
gene is controlled by two
regulators; one recruits
nucleosome modifiers and the
other recruits mediator
The HO gene is involved in the budding of yeast.
It has two activators, SWI5 and SBF.
alter the
nucleosome
Figure 17-15
3-3 Signal integration,
Cooperative binding of
activators at the human b-
interferon gene.
The human β-interferon gene is activated in cells
upon viral infection,Infection triggers three
activators,
NFκB,IRF,
and Jun/ATF,
They bind
cooperatively
to sites within
an enhancer,
form a
structure
called
enhanceosome.
Figure 17-16
3-4 Combinatory control lies at
the hear of the complexity and
diversity of eukaryotes
There is extensive combinatorial control in
eukaryotes.
In complex multicellular organisms,combinatorial
control involves many more regulators and genes
than shown above,and repressors as well as
activators can be involved,
Four
signals
Three
signals
Figure 17-17
3-5 Combinatory control of the
mating-type genes from S,
cerevisiae (啤酒酵母 )
The yeast S.cerevisiae exists in three
forms,two haploid cells of different
mating types－ a and a － and the diploid
formed when an a and an a cell mate
and fuse.
Cells of the two mating types differ
because they express different sets of
genes, a specific genes and a specific
genes.
a cell make the regulatory protein a1,
a cell make the protein a1 and a2.
A fourth regulator protein Mcm1 is
also involved in regulatory the mating-
type specific genes and is present in
both cell types.
How do these regulators work together
to keep a cell in its own type?
Control of cell-type specific genes in yeast
Figure 17-18
Ⅳ
Transcriptional
Repressors
In eukaryotes,repressors don’t work
by binding to sites that overlap the
promoter and thus block binding of
polymerase,but most common work by
recruiting nucleosome modifiers.
For example,histone deacetylases
repress transcription by removing
actetyl groups from the tails of histone.
Ways in
which
eukaryotic
repressor
Work
a and b
Figure 17-19
Ways in
which
eukaryotic
repressor
Work
c and d
Silencing
In the presence of glucose,Mig1 binds
a site between the USAG and the GAL1
promoter,By recruiting the Tup1
repressing complex,Mig1 represses
expression of GAL1,
A specific example,Repression of
the GAL1 gene in yeast
Ⅴ Signal Transduction
and
the Control of
Transcriptional Regulators
5-1 Signals are often
communicated to
transcriptional regulators
through signal transduction
pathway
Signals refers to initiating ligand (can
be sugar or protein or others),or just
refers to,information”.
There are various ways that signals
are detected by a cell and
communicated to a gene,But they are
often communicated to transcriptional
regulators through signal transduction
Pathway,in which the initiating ligand is
detected by a specific cell surface
receptor.
In a signal transduction pathway:
initiating ligand binds to an
extracellular domain of a specific cell
surface receptor this binding
bring an allosteric change in the
intracellular domain of receptor
the signal is relayed to the relevant
transcriptional regulator often
through a cascade of kinases.
5-2 Signals control the
activities of eukaryotic
transcriptional regulators in a
variety of ways
a,The STAT pathway
b,The MAP kinase pathway
Once a signal has been communicated,
directly or indirectly,to a transcriptional
regulator,how does it control the
activity of that regulator?
In eukaryotes,transcriptional regulators
are not typically controlled at the level
of DNA binding,They are usually
controlled in one of two basic ways,
Unmasking an activating region
Transport in or out of the nucleus
Activator Gal4 is regulated by masking protein Gal80
The signalling ligand causes activators (or
repressors) to move to the nucleus where
they act from cytoplasm.
5-3 Activators and repressors
sometimes come in pieces.
For example,the DNA binding domain
and activating region can be on
different polypeptides,same of an
activator
In addition,the nature of the protein
complexes forming on DNA
determines whether the DNA-binding
protein activates or represses nearby
genes,For example,the
glucocorticoid receptor (GR).
Ⅵ Gene,Silencing”
by
Modification of
Histones and DNA
Gene,silencing” is a position effect－ a
gene is silenced because of where it
is located,not in response to a
specific environmental signal.
The most common form of silencing is
associated with a dense form of
chromatin called heterochromatin,It
is frequently associated with
particular regions of the chromosome,
notably the telomeres,and the
centromeres.
6-1 Silencing in yeast is
mediated by deacetylation ane
methylation of the histones
The telomeres,the silent mating-type locus,
and the rDNA genes are all,silent” regions in
S.cerevisiae.
Three genes encoding regulators of silencing,
SIR2,3,and 4 have been found (SIR stand
for silent information regulator).
Silencing at the yeast telomere
6-1 Histone modification and
the histone code hypothesis
A histone code exists?
According to this idea,different
patterns of modification on histone tails
can be,read” to mean different things,
The,meaning” would be the result of
the direct effects of these
modifications on chromatin density and
form,
But in addition,the particular pattern
of modifications at any given location
would recruit specific proteins.
Transcription can also be silenced by
methylation of DNA by enzymes
called DNA methylases.
This kind of silencing is not found in
yeast but is common in mammalian
cells.
Methylation of DNA sequence can
inhibit binding of proteins,including
the transcriptional machinery,and
thereby block gene expression.
Switching a gene off,
A mammalian gene marked by methylation
of nearby DNA sequence
recognized by DNA-binding proteins
recruit histone decetylases and histone
methylases
modify nearby chromatin
This gene is completely off.
Switching a gene off
Figure 17-24
DNA methylation lies at the heart of a
phenomenon called imprinting,
Two examples,Human H19 and Igf2 genes.
Here an enhancer and an insulator are critical.
Patterns of gene expression must
sometimes be inherited,These
may remain for many cell generations,
even if the signal that induced them is
present only fleetingly,
This inheritance of gene expression
patterns is called epigenetic regulation.
Maintenance of a phage λlysogen,can
be described as an example.
λlysogens and the epigentic switchBox 3
Lysogenic gene expression is established in an
infected cell in response to poor growth
conditions,Then the lysogenic state will remain
through cell
division in
both cells,
This is
resulted from
a two-step
strategy for
repressor
synthesis.
How?
Nucleosome and DNA modifications
can provide the basis for epigenetic
inheritance.
DNA methylation is even more reliably
inherited,but far more efficiently is
the so-called maintenance methylases
modify hemimethylated DNA－ the very
substrate provided by replication of
fully methylated DNA.
Patterns of DNA methylation can be
maintained through cell division
Ⅶ Eukaryotic
Gene Regulation
at Steps
after
Transcription Initiation
In eukaryotic cells,some regulational
proteins aim at elongation.
At some genes there are sequence
downstream of the promoter that
cause pausing or stalling of the
polymerase soon after initiation.
At those genes,the presence or
absence of certain elongation
factors greatly influences the level
at which the gene is expressed.
Two examples, HSP70 gene and HIV
HIV genome
?
Many individual eukaryotic genes have exons
interrupted by introns,So when the whole gene
is transcribed,mRNA need to be spliced.
In some cases a given precursor mRNA
can be spliced in alternative ways to
produce different mRNAs that encode
different protein products.
The regulation of alternative splicing
works in a manner reminisencent, the
splicing machinery binds to splice sites
and carries out the splicing reaction.
The sex of a fly is determined by the
ratio of X chromosomes to autosomes,
This ratio is initially measured at the
level of transcription using two
activators SisA and SisB,The genes
encoding these regulators are both on
the X chromosome.
Sxl, Sex-lethal
Dpn,Deadpan
Early transcriptional regulation of Sxl in
male and female flies
A cascade
of
alternative
splicing
events
determines
the sex
of a fly
Gcn4 is a yeast transcriptional activator
that regulates the expression of genes
encoding enzymes that direct amino acid
biosynthesis,
The mRNA encoding the Gcn4 protein
contains four small open reading frames
(called uORFs) upstream of the coding
sequence for Gcn4.
Although it is a activator,Gcn4 is itself
regulated at the level of translation,
In the presence of low levels of amino
acids,the Gcn4 mRNA is translated
(and so the biosynthetic are
expressed),
In the presence of high levels,it is
not translated.
How is this regulation achieved?
high levels
of amino
acids,
the Gcn4
mRNA
is not
translated
low levels
of amino
acids,
the Gcn4
mRNA is
translated
Ⅷ RNAs
in
Gene Regulation
8-1 Double-standed RNA
inhibits expression of genes
homologous to that RNA
8-2 Short interfering RNA
(siRNAs) are produced from
dsRNA and direct machinery
that switch off genes in various
way
8-3 MicroRNA control the
expression of some genes
during development.
Short RNAs can direct repression of
genes with homology to those short
RNAs,
This repression,called RNA
interference (RNAi),can manifest as
translational inhibition of the mRNA,
destruction of the mRNA or
transcriptional silencing of the
promoter that directs expression of
that mRNA.
The discovery that simply introducing
double-strand RNA (dsRNA) into a cell
can repress genes containing sequence
identical to (or very similar to) that
dsRNA was remarkable in 1998 when
it was reported.
A similar effect is seen in many other
organisms in both animals and plants.
How dsRNA can switch off expression
of a gene?
Dicer is an RNAseⅢ -like enzyme that
recognizes and digests long dsRNA,The
products are short double-stranded fragments.
These short RNAs (or short interfering RNAs,
siRNAs) inhibit expression of a homologous gene
in three ways,
Trigger destruction of its mRNA
Inhibit translation of its mRNA
Induce chromatin modifications within the
promoter that silence the gene
That machinery includes a complex called RISC
(RNA-induced silencing complex).
RNA
silencing
RNAi silencing is extreme efficiency.
Very small amounts of dsRNA are
enough to induce complete shutdown
of target genes.
Why the effect is so strong?
It might involve an RNA-dependent
RNA polymerase which is required in
many cases of RNAi.
There is another class of naturally
occurring RNAs,called microRNAs
(miRNAs),that direct repression of
genes in plants and worms.
Often these miRNAs are expressed in
developmentally regulated patterns.
The mechanism of RNAi may have
evolved originally to protect cells from
any infectious,or otherwise disruptive,
element that employs a dsRNA
intermediate in its replicative cycle.
Now RNAi has been adapted for use as
a powerful experimental technique
allowing specific genes to be switched
off in any of many organisms.
Summary,
There are several complexities in the
organization and transcription of
eukaryotic genes not found in bacteria,
Nucleosomes and their modification
Many regulators and larger distances
The elaborate transcriptional machinery
Pay attention to these differences,and tell
them in details by yourself.
Do you know these conceptions?
Promoter
Regulator binding site
Regulatory sequence
Enhancer
Insulator
Reporter gene
Gene silencing
Critical Thinking Exercises
1,Compare the mechanisms used by zinc-binding,leucine zipper,
and HLH motifs to bind DNA,What is the role of zinc in the
zinc-binding domain?
2,Describe the physical characteristics of a typical transcriptional
activation region,How are these characteristics thought to
reflect the mechanism of activation? If a novel transcription
factor is identified,and you would like to locate the domain
within the protein that is responsible for activation,how would
you do this?
3,You transform a population of cells with a transgene,and
isolate a cell line that has integrated the gene into its genome,
but in which the gene is not expressed,Speculate as to what
may be preventing the gene from being expressed,Describe
two ways to test this possibility,
4,To determine how an activator contributes to the formation
of the holoenzyme at your favorite promoter,design an
experimental strategy to tell you which components are
directly recruited by the activator,and which of these
activator-mediated recruitment steps are required for
transcriptional activation,How could you test whether the
role of the activator stops with the holoenzyme assembly,
or if it also induces allosteric changes within the DNA or the
holoenzyme?
5,You isolate a mutant strain of mice that grow at an
unusually fast rate,perform a blood test on the mice,and
find that they have elevated levels of insulin-like growth
factor,The phenotype is the result of a mutation in a region
of the genome containing a gene encoding a DNA
methylase,What kind of mutation is causing the rapid
growth of these mice?
