Transcript N1 Eukaryotic transcription factors

Section N – Regulation of
transcription in eukaryotes
Contents
N1 Eukaryotic transcription factors
Transcription factor domain structure, DNAbinding domains, Dimerization domains,
Transcription activation domains, Repressor
domains, Targets for transcriptional regulation
N2 Eukaryotic of transcriptional
regulation
Constitutive transcription factors:SP1, Hormonal
regulation: steroid hormone receptors, Regulation
by phosphorylation: STAR proteins, Transcription
elongation: HIV Tat, Cell determination: myoD,
Embrynic development: homeodomain proteins
N1 Eukaryotic transcription factors —
Transcription factor domain structure
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
N1 Eukaryotic transcription factors —
DNA- binding domains
1. The helix-turn-helix domain
2. The zinc finger domain
3. The basic domain
1. The helix-turn-helix domain
Examples of Helix-turn-helix domains
1). Homeodomain: encoded by a sequence
called the homeobox, containing a 60amino-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.
2. 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
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.
C4 zinc finger: zinc ion is
coordinated by 4 cysteine
residues.
②
Example: steriod hormone receptor
transcription factors (N2) consisting of
homo- or hetero-dimers, in which each
monomer contains two C4 zinc finger.
N1 Eukaryotic transcription factors —
Dimerization domains
• Leucine zippers
• The helix-loop-helix domain
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 .
• 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
• 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 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 Eukaryotic transcription factors —
Transcription activation domains
1. Acidic activation domains
2. Glutamine-rich domains
3. Proline-rich domains
Acidic activation domains
• Also called “acid blobs” or
“negative noodles”
• Rich in acidic amino acids
• Exists in many transciption
activation domains
1. yeast Gcn4 and Gal4,
2. mammalian glucocorticoid
receptor
3. 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 Eukaryotic transcription factors —
Repressor domains
• 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).
• 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
N1 Eukaryotic transcription factors —
Targets for transcriptional regulation
• 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.
N2 Eukaryotic of transcriptional regulation —
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 Eukaryotic of transcriptional regulation —
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.
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 Eukaryotic of transcriptional regulation —
Regulation by phosphorylation:
STAR 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).
1. Unphosphorylated STAT1α protein:
exists as a monomer in the cell
cytoplasm and has no
transcriptional activity.
2. 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
N2 Eukaryotic of transcriptional regulation —
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.
• 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
N2 Eukaryotic of transcriptional regulation —
Cell determination: myoD
• 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 musclespecific 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 Eukaryotic of transcriptional regulation —
Embrynic 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.
Multiple choice questions
1.
A
B
C
D
Which two of the following statements about transcription factors are true?
the helix-turn-helix domain is a transcriptional activation domain.
dimerization of transcription factors occurs through the basic domain.
leucine zippers bind to DNA.
it is often possible to get functional transcription factors when DNA binding domains
and activation domains from separate transcription factors are fused together.
E the same domain of a transcription factor can act both as a repressor and as an
activation domain.
2． Which two of the following statements about transcriptional regulation are
false?
A SP1 contains two adivation domains.
B steroid hormones regulate transcription through binding to cell surface receptors.
C phosphorylation of Stat1α leads to its migration from the cytoplasm to the nucleus.
D HIV Tat regulates RNA Pol II phosphorylation and processivity.
E the MyoD protein can form heterodimers with a set of other HLH transcription factors.
F the homeobox is a conserved DNA binding domain.
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