Download Section N – Regulation of transcription in eukaryotes

•Molecular Biology Course
Section N
Regulation of
transcription in
eukaryotes
N1 Eukaryotic Transcription
Factors
1. Transcription factor domain structure:
(DNA-binding, dimerization,
transcription activation, repressor)
2. Targets for transcriptional regulation
N2 Examples of
transcriptional regulation
SP1, hormonal regulation, phosphorylation
of STAT proteins, HIV Tat, myoD
homeodomain proteins
Section N: Regulation of transcription in eukaryotes
N1 Eukaryotic Transcription Factors

Transcription factor domain
structure (link)
1.
DNA-binding domains
Dimerization domains
Transcription activation domains
Repressor domains
2.
3.
4.

Targets for transcriptional
regulation (link)
Transcription of a single gene may
be regulated by many different
factors interacting with regulatory
elements upstream or downstream
of the transcribed sequence.
Start site
Gene X
+1
Regulatory elements
to bind transcription factors
Example:
The metallothionein (MT, 金属硫蛋白) gene
The metallothionein protein protects the cell against
excess concentrations of heavy metals, by binding the
metal and removing it from the cell. The gene is
expressed at a basal level, but is induced to greater levels
of expression by heavy metal ions (such as cadmium) or
by glucocorticoids (糖皮质素).
Common features of transcription factors
1.
2.
bind specifically to some DNA sites:
specific motifs in promoters, upstream
regulatory elements (UREs) or enhancer
regions. Some factors modulate
transcription by protein-protein intracation
Activate/repress transcription.
Transcription factors – domain structure
Transcription factor Pdr1
- The
activity of a transcription factor can be
assigned to separate protein domains
activation
domains. (activity)
DNA-binding
domains. (activity)
dimerization
domains. Many transcription factors
occur as homo- or heterodimers, held together by
dimerization domains. (regulation)
ligand-binding
domains. Allowing regulation of
transcription factor activity by binding of an accessory
small molecule.The steroid hormone receptors are an
example containing all for of these types of domain.
(regulation)
Domain swap experiments
moving domains among proteins,
proving that domains can be
dissected into separate parts of the
proteins.
The experiment of fusing activation
domains of yeast transcription factors
Gal4 and Gcn4 into the bacterial LexA
repressor is described in your text
book. Transcription activation domains
are separable from their DNA binding
activity.
Another example: construction of new
proteins capable of binding to DNA
NLS: nucleus localization signal
N1-2:
DNA-binding domains
1.
2.
3.
The helix-turn-helix domain
The zinc finger domain
The basic domain
Return to menu…
The helix-turn-helix domain
Examples of Helix-turn-helix domains
1. Homeodomain: encoded by a
sequence called the homeobox,
containing a 60-amino-acid. In the
Antennapedia transcription factor of
Drosophila, this domain consists of
four α-helices in which helices Ⅱand
Ⅲ are at right angles to each other
and are separated by a characteristic
β-turn.
2. Bacteriophage DNA-binding proteins
such as the phage λ cro repressor, lac
and trp repressors, and cAMP
receptor protein, CRP.
The
recognition helix of the domain
structure lies partly in the major groove and
interacts with the DNA.
The
recognition helices of two
homeodomain factors Bicoid and
Antennapedia can be exchanged, and this
swaps their DNA-binding specificities.
The zinc finger domain

Zinc finger domain exists in
two forms.
1.
C2H2 zinc finger: a loop of 12 amino
acids anchored by two cysteine and
two histidine residues that
tetrahedrally co-ordinate a zinc ion.
This motif folds into a compact
structure comprising two β-strands
and one α-helix. The α-helix
containing conserved basic amino
acids binds in the major groove of
DNA (picture…picture2)
Examples:
(1) TFIIIA, the RNA Pol III
transcription factor, with C2H2 zinc
finger repeated 9 times.
(2) SP1, with 3 copies of C2H2 zinc
finger.
Usually, three or more C2H2 zinc
fingers are required for DNA
binding.
2. C4 zinc finger: zinc ion is
coordinated by 4 cysteine
residues.
Example: steriod hormone
receptor transcription factors (N2)
consisting of homo- or heterodimers, in which each monomer
contains two C4 zinc finger.
(picture…)
The basic domain
Rich in basic amino acid residues
 found in a number of DNA-binding
proteins
 generally associated with one or
other of two dimerization domains,
the leucine zipper or the helix-loophelix(HLH) motif, resulting in basic
leucine zipper (bZIP) or basic HLH
proteins. Dimerization of the proteins
brings together two basic domains
which can then interact with DNA.

N1-3: Dimerization
domains
Leucine
zippers
The helix-loop-helix
domain (HLH)
Leucine zippers
Leucine zipper proteins contain a
hydrophobic leucine residue at every
seventh position in a region that is
often at the C-terminal part of the
DNA-binding domain (picture.).
 These leucines are responsible for
dimerization through interaction
between the hydrophobic faces of the
α-helices. This interaction forms a
coiled-coil structure

bZIP (basic leucine zipper) transcription
factors: contain a basic DNA-binding
domain N-terminal to the leucine zipper.
The N-terminal basic domains of each helix
form a symmetrical structure in which each
basic domains lies along the DNA in
opposite direction, interacting with a
symmetrical DNA recognition site with the
zippered protein clamp (pic1..)
 The leucine zipper is also used as a
dimerization domain in proteins containing
DNA-binding domains other than the basic
domain, including some homeodomain
proteins.

The helix-loop-helix domain
(HLH)
The overall structure is similar to the
leucine zipper, except that a
nonhelical loop of polypeptide chain
separates two α-helices in each
monomeric protein.
 Hydrophobic residues on one side of
the C-terminal α-helix allow
dimerization.
 Example: MyoD (pic..) family of
proteins.

Similar
to leucine zipper, the HLH
motif is often found adjacent to a
basic domain that requires
dimerization for DNA binding.
Basic
HLH proteins and bZIP
proteins can form heterodimers
allowing much greater diversity and
complexity in the transcription factor
repertoire.
N1-4: Transcription
activation domains
Acidic
activation domains
Glutamine-rich domains
Proline-rich domains
Acidic activation domains



1.
2.
3.
Also called “acid blobs” or
“negative noodles”
Rich in acidic amino acids
Exists in many transciption
activation domains
yeast Gcn4 and Gal4,
mammalian glucocorticoid
receptor
herpes virus activator VP16
domains.
Glutamine-rich domains
 Rich
in glutamine
 the proportion of glutamine
residued seems to be more
important than overall structure.
 Exists in the general
transcription factor SP1.
Proline-rich domains
 Proline-rich
 continuous
run of proline
residues can activate
transcription
 Exists in transcription factors cjun, AP2 and Oct-2.
N1-5: Repressor domains

1.
2.
Repression of transcription may occur by
indirect interference with the function of
an activator. This may occur by:
Blocking the activator DNA-binding site
(as with prokaryotic repressors, wrong)
Formation of a non-DNA-binding complex
(e.g. the Id protein which blocks HLH
protein-DNA interactions, since it lacks a
DNA-binding domain, N2).
3. Masking of the activation domain without
preventing DNA binding (e.g. Gal80 masks
the activation domain of the yeast
transcription factor Gal4).
4. A specific domain of the repressor is
directly responsible for inhibition of
transcription. (e.g. prokaryotic repressors)
 e.g. A domain of the mammalian thyroid
hormone receptor can repress
transcription … (page 212 & 214).
Return to menu…
N1-6: Targets for
transcriptional regulation
(pic…)
1.
2.
3.
4.
chromatin structure;
interaction with TFIID through specific
TAFIIS;
interaction with TFIIB;
interaction or modulation of the TFIIH
complex activity leading to differential
posphorylation of the CTD of RNA Pol II.
 It
seems likely that different
activation domains may have
different targets, and almost any
component or stage in initiation
and transcription elongation
could be a target for regulation
resulting in multistage regulation
of transcription.Return to menu…
Section N: Regulation of transcription in eukaryotes
N2 Examples of
transcriptional regulation
1.
Constitutive transcription
factors:SP1
2.
Hormonal regulation:steroid
hormone receptors
3.
Regulation by phosphorylation:STAT
proteins
4.
Transcription elongation:HIV Tat
5.
Cell determination:myoD
6.
Embryonic
development:homeodomain proteins
N2-1: Constitutive
transcription factors:SP1
binds to a GC-rich sequence with the
consensus sequence GGGCGG.
 binding site is in the promoter of many
housekeeping genes
 It is a constitutive transcription factor
present in all cell types.
 contains three zinc finger motifs and two
glutamine-rich activation domains
interacting with TAFII110, thus regulating
the basal transcription complex.

N2-2: Hormonal
regulation:steroid hormone
receptors
Many transcription factors are
activated by hormones which are
secreted by one cell type and
transmit a signal to a different cell
type.
 steroid hormones: lipid soluble and
can diffuse through cell membranes
to interact with transcription factors
called steroid hormone receptors.



1.
2.
3.
In the absence of steroid hormone,
the receptor is bound to an inhibitor,
and located in the cytoplasm (picture).
In the presence of steroid hormone,
the hormone binds to the receptor
and releases the receptor from the
inhibitor,
receptor dimerization and
translocation to the nucleus.
receptor interaction its specific DNAbinding sequence (response element)
via its DNA-binding domain,
activating the target gene.
Steroid
hormones involving
important hormone receptors:
glucocorticoid (糖皮质激素）,
estrogen (雌激素), retinoic acid （视
黄酸）and thyroid hormone （甲状
腺激素）receptors.
Please noted that the above model is
not true for all these hormone receptors
Thyroid
hormone receptor is a DNAbound repressor in the absence of
hormone, which converted to a
transcriptional activator.
N2-3: Regulation by
phosphorylation: STAT
proteins

For hormones that do not diffuse into the cell.
 The hormones binds to cell-surface receptors
and pass a signal to proteins within the cell
through signal transduction.
 Signal transduction often involves protein
phosphorylation.
Example: Interferon-γ induces phosphorylation of
a transcription factor called STAT1α through
activation of the intracellular kinase called
Janus activated kinase(JAK). go on...
1.
2.
Unphosphorylated STAT1α protein:
exists as a monomer in the cell
cytoplasm and has no
transcriptional activity.
Phosphorylated STAT1α at a specific
tyrosine residue forms a homodimer
which moves into the nucleus to
activate the expression of target
genes whose promoter regions
contain a consensus DNA-binding
motif (picture…pic3..)
N2-4: Transcription
elongation:HIV Tat
Human immunodeficiency virus
(HIV)(pic…) encodes an activator
protein called Tat, which is required for
productive HIV gene expression(pic..).
 Tat binds to an RNA stem-loop structure
called TAR, which is present in the 5’UTR of all HIV RNAs just after the HIV
transcription start site, to regulate the
level of transcription elongation.

In the absence of Tat, the HIV
transcripts terminate prematurely due to
poor processivity of the RNA Pol Ⅱ
transcription complex.
 Tat binds to TAR on one transcript in a
complex together with cellular RNAbinding factors. This protein-RNA
complex may loop backwards and
interact with the new transcription
initiation complex which is assembled at
the promoter. go on...


This interaction may result in the
activation of the kinase activity of TFIIH,
leading to phosphorylation of the
carboxyl-terminal domain (CTD) of RNA
PolⅡ, making the polymerase a
processive enzyme to read through the
HIV transcription unit, leading to the
productive synthesis of HIV proteins
(picture..)
N2-5: Cell determination:myoD
(pic1..pic2..)

myoD was identified as a gene to regulate gene
expression in cell determination, commanding
cells to form muscle.
 MyoD protein has been shown to activate
muscle-specific gene expression directly.
Overexpression of myoD can turn fibroblasts into
muscle-like cells which express muscle-specific
genes and resemble myotomes.
 myoD also activates expression of p21waf1/cip1
expression, a small molecule inhibitor of CDKs,
causing cells arrested at the G1-phase of the cell
cycle which is characteristic of differentiated
cells. .

Four genes,myoD,myogenin, myf5 and
mrf4 have been shown to have the ability
to convert fibroblasts into muscle. The
encoded proteins are all members of the
helix-loop-helix (HLH for dimerization)
transcription factor family.
 These proteins are regulated by an
inhibitor called Id that lacks a DNA-binding
domain, but contains the HLH dimerization
domain. Id protein can bind to MyoD and
related proteins, but the resulting
heterodimers cannot bind DNA, and hence
cannot regulate transcription
N2-6: Embryonic development:
homeodomain proteins

The homeobox is a conserved DNA sequence
which encodes the helix-turn-helix DNA binding
protein structure called the homeodomain.
 Homeotic genes of Drosophila are responsible
for the correct specification of body parts. For
example, mutation of one of these genes,
Antennapedia, causes the fly to form a leg
where the antenna should be.
 conserved between a wide range of eukaryotes.
 important in mammalian development.
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1. TFIID:
• multiprotein complex
including TBP, other
proteins are known
as TAFIIs
• TBP is the only
protein binds to
TATA box
3. TFIIB &
RNA Pol
binding
• binds to TFIID
•Binds to RNA
Pol with TFIIF
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5. phosphorylation of the polymerase CTD
by TFIIH
Formation of a processive RNA polymerase complex and
allows the RNA Pol to leave the promoter region.
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HIV genome
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