Transcript What is trans-acting factor?

Chapter 13
Gene Regulation
in Eukaryotes
Eukaryotic gene regulation occurs
at several levels
1- Control at DNA level by DNA methylation

Small percentages of newly synthesized DNAs (~3% in
mammals) are chemically modified by methylation.

Methylation occurs most often in symmetrical CG
sequences.

Transcriptionally active genes possess significantly lower
levels of methylated DNA than inactive genes.

Methylation results in a human disease called fragile X
syndrome; FMR-1 gene is silenced by methylation.
2- Control at DNA level by Histone
modifications(Chromatin Remodeling)
•
Acetylation (乙酰化)by
histone acetyl transferases
(HATs) and coactivators leads
to euchromatin formation; p53
acetylation
•
Methylation by HDACs （去
乙酰化酶）and corepressors
leads to heterochromatin
formation . Rb-E2F
3-Control at DNA level by gene amplification
Repeated rounds of DNA replication yield multiple
copies of a particular chromosomal region.
4- Control at transcription initiation
By using different sequences (promoter,
enhancer or silencer sequences) and factors,
the rate of transcription of a gene is controlled
gene control region for gene X
5- Control at mRNA splicing
(alternate splicing)
(four exons)
Calcitonin
gene-related
peptide
32 amino acids
Reduces bone resorption
37 amino acids
Vasodilator
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6- Control at mRNA stability
• Messenger RNA longevity can be influenced by
several factors.
• Poly(A) tails seem to stabilize mRNAs.
• The sequence of the 3’untranslated region (3’UTR)
preceding a poly(A) tail also seems to affect mRNA
stability.
• Several short-lived mRNAs have the sequence
AUUUA repeated several times in their 3’untranslated
regions.
6- Control at mRNA stability
• When this sequence is artificially transferred to the
3’untranslated region of more stable mRNAs, they, too,
become unstable.
• Chemical factors, such as hormones, may also affect
mRNA stablility.
• In the toad Xenopus laevis(非洲爪蟾) , the
vitellogenin gene(卵黄生成素) is transcriptionally
activated by the steroid hormone estrogen(类固醇激
素 ) . However, in addition to inducing transcription of
this gene, estrogen also increases the longevity of its
mRNA.
6- Control at mRNA stability
• Recent research has revealed that the stability
of mRNAs and the translation of mRNAs into
polypeptides are also regulated by small,
noncoding RNA molecules called microRNA
(miRNAs).
7- Control at initiation of translation
5’
UTR
AUG
3’
UAA UTR
Specific sequences make specific secondary structures
Specific protein factors bind to these secondary structures
8-Regulation by protein stability
•Ubiquitin-dependent proteolysis.
•Protein molecule is tagged for degradation by attachment of a 20 kDa
protein, ubiquitin
ATP
NH2
NH2
+
Doomed protein
molecule
CO NH
COOH
ubiquitin
protein ligase
CO NH
26S
proteasome
蛋白酶体系统(ubiquitin-proteasome system(UPS))主要由泛
素激活酶(E1)、泛素交联酶(E2)、泛素连接酶(E3)和26S蛋白酶体
组成,是降解细胞内蛋白质的主要途径
对于许多细胞进程，包括细胞周期、基因表达的调控、氧化应激反应等，
都是必不可少的。2004年诺贝尔化学奖.
Similarity of regulation between
eukaryotes and prokaryote
1.Principles are the same:
signals (信号),
activators and repressors (激活蛋白和阻遏蛋白)
recruitment and allostery, cooperative binding (招募，
异构和协同结合)
2. The gene expression steps subjected to regulation are
similar, and the initiation of transcription is the most
pervasively regulated step.
Difference in regulation between
eukaryotes and prokaryote*****
1.
2.
3.
4.
Pre-mRNA splicing adds an important step for
regulation. (mRNA前体的剪接)
The eukaryotic transcriptional machinery is more
elaborate than its bacterial counterpart. (真核转录机
器更复杂)
Nucleosomes and their modifiers influence access to
genes. (核小体及其修饰体)
Many eukaryotic genes have more regulatory binding
sites and are controlled by more regulatory proteins
than are bacterial genes. (真核基因有更多结合位点)
A lot more regulator bindings sites in
multicellular organisms reflects the
more extensive signal integration
Bacteria
Yeast
Human
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Cis-acting element

Promoter
 Core promoter
 in eukaryote: TATA-box, Initiator (Inr)
 in prokaryote: -10 region, Inr
 Proximal elements of promoter
 in prokaryote: -35 region
 in eukaryote: CAAT-box, GC-box
UPE: upstream promoter element
UAS: upstream activating sequence

Terminator (终止子)：A DNA sequence just
downstream of the coding segment of a gene, which is
recognized by RNA polymerase as a signal to stop
transcription.
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Enhancer (激活元件) : 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.
Silencer （沉默子）A DNA sequence that helps to
reduce or shut off the expression of a nearby gene.
Insulators (绝缘子) or boundary elements (临界元件)
are regulatory sequences between enhancers and
promoters. They block activation of a linked promoter by
activator bound at the enhancer, and therefore ensure
activators work discriminately.
What is trans-acting factor?
Usually they are proteins, that bind
to the cis-acting elements to control
gene expression.
These trans-acting factors can control
gene expression in several ways:
 may be expressed in a specific tissue
 may be expressed at specific time in
development
 may be required for protein modification
 may be activated by ligand binding
(1) RNA polymerase
 prokaryotic RNA Pol
 eukaryotic RNA Pol
(2) Transcription factors
 Basal/general TFs
 Specific TFs
(3) Domains of trans-acting factors
 DNA binding domain DBD
DNA结合结构域
 transcription activating domain
转录活化结构域
一、真核的转录激活蛋白的结构特征
The structure features of the
eukaryotic transcription activators
Topic 1: Conserved
Mechanisms of
Transcriptional Regulation
from Yeast (酵母) to
Mammals (哺乳动物)
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.
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1. Eukaryotic activators (真核激活蛋白) have
separate DNA binding and activating
functions, which are very often on separate
domains of the protein.*****
Gal4 bound to its site on DNA
Eukaryotic activators---Example 1: Gal4*****
 Gal4 is the most studied eukaryotic activator
 Gal4 activates transcription of the galactose genes in
the yeast S. cerevisae.
 Gal4 binds to four sites (UASG) upstream of GAL1(5'CGGRNNRCYNYNYNCNCCG-3' ), and activates transcription
1,000-fold in the presence of galactose
The regulatory sequences of the Yeast GAL1 gene.
Experimental evidences showing that Gal4 contains
separate DNA binding and activating domains.
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.
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Box1 The two hybrid Assay (双杂交) to study proteinprotein interaction and identify proteins interacting with a
known protein in cells*****
Fuse protein A and protein B
genes to the DNA binding
domain and activating region of
Gal4, respectively.
Produce fusion proteins
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2. Eukaryotic regulators use a range of DNA
binding domains, but DNA recognition
involves the same principles as found in
bacteria. *****
Transcription factor motifs




Helix-turn-helix ( HTH) (螺旋-转角-螺旋)
Zinc finger （锌指）and zinc cluster
Leucine zipper motif（亮氨酸拉链）
Helix-Loop-Helix proteins （螺旋-突环-螺旋）: basic zipper
and HLH proteins
HTH (helix-turn-helix)
α-helix (N-terminus)----specific
α-helix (C-terminus)----non-specific
Bacterial regulatory proteins
• Most use the helix-turn-helix (HTH：旋转-转角-旋转）
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. In addition to form homodimers (同源二聚体),
some form heterodimers (异源二聚体) to
recognize DNA, extending the range of DNAbinding specificity.
Zinc containing DNA-binding domains (锌指
结构域): Zinc finger proteins (TFIIIA) and
Zinc cluster domain (Gal4)
Leucine Zipper Motif (亮氨酸拉链基序) : The
Motif combines dimerization and DNAbinding surfaces within a single structural
unit.
Helix-Loop-Helix motif
myogenic factor:生肌调节蛋白是
一种转录因子。
3. Activating regions (激活区域) are
not well-defined structures***
The activating regions are grouped on
the basis of amino acids content.
 Acidic activation region (酸性激活区域):
contain both critical acidic amino acids
and hydrophobic aa. yeast Gal4
 Glutamine-rich region (谷氨酰胺富集区):
mammalian activator SP1
 Proline-rich region (脯氨酸富集区):
mammalian activator CTF1
二、真核转录激活蛋白的招募调控方式和远距调控特征
Activation of the eukaryotic transcription
by recruitment & Activation at a distance
Topic 2: Recruitment of
Protein Complexes
to Genes by
Eukaryotic Activators
Eukaryotic activators (真核激活蛋白)
also work by recruiting (招募) as in
bacteria, but recruit polymerase
indirectly in two ways:
1. Interacting with parts of the
transcription machinery.
2. Recruiting nucleosome modifiers
that alter chromatin in the vicinity
of a gene.
1. Activators recruit the transcription
machinery to the gene.
The eukaryotic transcriptional machinery contains
polymerase and numerous proteins being organized to
several complexes, such as the Mediator and the TFⅡD
complex. Activators interact with one or more of these
complexes and recruit them to the gene.
Chromatin Immuno-precipitation (ChIP) (染色质免
疫共沉淀) to visualize where a given protein
(activator) is bound in the genome of a living
cell*****.)
2. Activators also recruit modifiers that
help the transcription machinery bind
at the promoter
Two types of Nucleosome modifiers :
Those add chemical groups to the tails
of histones (在组蛋白尾上加化学基团), such
as histone acetyl transferases (HATs)
Those remodel the nucleosomes (重塑核
小体), such as the ATP-dependent
activity of SWI/SNF.
How the nucleosome modification
help activate a gene?*****
1.
“Loosen” the chromatin structure by
chromosome remodeling and histone
modification such as acetylation,
which uncover DNA-binding sites
that would otherwise remain
inaccessible within the nucleosome.
Local
alterations
in chromatin
structure
directed
by activators
Activators,
capable of binding
to their sites on
DNA within a
nucleosome are
shown bound
upstream of a
promoter that
(a) The activator is shown recruiting (b)The activator recruits a
is inaccessible a histone acetylase. That enzyme
nucleosome remodeller, which
within chromain. adds acetyl groups to residues within alters the structure of
the histone tails. This alters the
nucleosomes around the
packing of the nucleosomes
promoter, rendering it
somewhat, and also creates binding accessible and capable of
sites for proteins carrying the
binding the transcription
appropriate recognition domains.
machinery.
3. Action at a distance: loops and
insulators
Specific cis-acting elements called
insulators (绝缘子) control the
actions of activators, preventing the
activating the non-specific genes
a) A promoter activated
by activators bound
to an enhancer.
b) An insulator is
placed between the
enhancer and the
promoter. When
bound by appropriate
insulator- binding
proteins, activation
of the promoter by the
enhancer is blocked,
despite activators
binding to the enhancer.
c) The activator can
activate another
promoter nearby.
d) The original promoter
can be activated by
another enhancer
placed downstream.
Insulators block activation by enhancers.
Transcriptional Silencing (转录沉默)
Transcriptional Silencing is a specialized 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。
4 Appropriate regulation of some groups of
genes requires locus control region (LCR).
1.
2.
Human and mouse globin genes are clustered
in genome and differently expressed at
different stages of development
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 binds regulatory proteins
that cause the chromatin structure to “open
up”, allowing access to the array of
regulators that control expression of the
individual genes in a defined order.
(a)The human
globin genes and the
LCR that ensures
their ordered
expression.
(b) The globin genes
from mice, which are
also regulated by an
LCR.
(C) The HoxD gene
cluster from the
mouse controlled by
an element called
the GCR which like
the LCRs appears to
impose ordered
expression on the
gene cluster.
Regulation by LCRs
三、真核转录阻遏蛋白（或抑制蛋白）及其调控
Topic 3: Transcriptional
Repressor & its regulation
In eukaryotes, most repressors do not
repress transcription by binding to sites
that overlap with the promoter and thus
block binding of polymerase. (Bacteria
often do so)
Commonly, eukaryotic repressors recruit
nucleosome modifiers that compact the
nucleosome or remove the groups
recognized by the transcriptional
machinery [contrast to the activator
recruited nucleosome modifiers, histone
deacetylases (组蛋白去乙酰化酶) removing
the acetyl groups]. Some modifier adds
methyl groups to the histone tails, which
frequently repress the transcription.
This modification causes transcriptional
silencing.
Three other ways in which an eukaryotic
repressor works include:
(1) Competes with the activator for an
overlapped binding site.
(2) Binds to a site different from that of
the activator, but physically interacts
with an activator and thus block its
activating region.
(3) Binds to a site upstream of the
promoter, physically interacts with the
transcription machinery at the promoter
to inhibit transcription initiation.
Competes for the
activator binding
Inhibits the function
of the activator.
Ways in which eukaryotic repressor work
Binds to the
transcription
machinery
Recruits nucleosome
modifiers (most common***)
A specific example: Repression of
the GAL1 gene in yeast
In the presence of glucose, Mig1 binds to a
site between the USAG and the GAL1 promoter,
and recruits the Tup1 repressing complex.
Tup1 recruits histone deacetylases, and also
directly interacts with the transcription
machinery to repress transcription.
四、基于真核转录调控的前沿学科：信号传导
Signal transduction---A life science
frontier centered on the eukaryotic
transcriptional regulation.
Topic 4: Signal
Transduction (信号传导) and
the Control of
Transcriptional Regulators
1. Signals are often communicated to
transcriptional regulators through
signal transduction pathway
信号经常通过信号传导途径被运输到转录调节蛋白
Environmental Signals/Information (信号)
1. Small molecules such as sugar,
histamine (组胺).
2. Proteins released by one cell and
received by another.
In eukaryotic cells, most signals are
communicated to genes through signal
transduction pathway (indirect), in which
the initiating ligand is detected by a
specific cell surface receptor.
Signal transduction pathway***
1. The initial ligand (“signal”) binds to an
extracellular domain of a specific cell
surface receptor
2. The signal is thus communicated to the
intracellular domain of receptor (via an
allosteric change or dimerization )
3*. The signal is then relayed (分程传递) to
the relevant transcriptional regulator.
4. The transcriptional regulator control
the target gene expression.



JAK activation occurs upon ligand-mediated
receptor multimerization because two JAKs are
brought into close proximity, allowing transphosphorylation.
The activated JAKs subsequently phosphorylate
additional targets, including both the receptors and
the major substrates, STATs.
STATs are latent transcription factors that reside in
the cytoplasm until activated.
MAP kinases are activated within the protein kinase cascades called “MAPK cascade”.
Each one consists of three enzymes, MAP kinase, MAP kinase kinase (MKK, MEK, or
MAP2K) and MAP kinase kinase kinase (MKKK, MEKK or MAP3K) that are activated in
series.
A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine
and threonine residues, and this MAP2K activates a MAP kinase through phosphorylation
on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2).
2. Signals control the activities of
eukaryotic transcriptional regulators
in a variety of ways.
信号通过不同方式控制转录调节蛋白的活性
Mechanism 1: unmasking an activating
region:
(1)
(2)
(3)
A conformational change to reveal the
previously buried activating region.
Releasing of the previously bound masking
protein. Example: the activator Gal4 is
controlled by the masking Gal80).
Some masking proteins not only block the
activating region of an activator but also
recruit a deacetylase enzyme to repress the
target genes. Example: Rb represses the
function of the mammalian transcription
activator E2F in this way. Phosphorylation of
Rb releases E2F to activate the target gene
expression***.
Activator Gal4 is regulated by a
masking protein Gal80***
Gal4
Mechanism 2: Transport into and out of
the nucleus:
When not active, many activators and
repressors are held in the cytoplasm. The
signaling ligand causes them to move into the
nucleus where they activate transcription
Other Mechanisms #1: A cascade of
kinases that ultimately cause the
phosphorylation of regulator in nucleus
(new) (Fig.19-4a).
Other Mechanisms #2: The activated
receptor is cleaved by cellular proteases
(蛋白酶), and the c-terminal portion of
the receptor enters the nuclease and
activates the regulator (new):(Fig.19-4c).