Download Activators of eukaryotic transcription initiation

Activators of eukaryotic transcription initiation
Proteins which activate transcription initiation by RNA
polymerase II and III are called transcription factors.
These should not be confused with GTFs that play a
more central role in the assembly of the preinitiation
complex.
Traditional view – transcription factors are sequencespecific DNA-binding proteins.
Some recognize upstream promoter elements and influence
the transcription only at the promoter to which these
elements are attached.
Others target sites within enhancers and influence
transcription of several genes at once.
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From T. Brown, Genomes
Traditional view:
Whether bound to an upstream promoter element or to a
more distant enhancer, activator stimulates formation
of preinitiation complex
(i) by making contact with it,
(ii) directly or via another protein, sometimes achieving
this
(iii) by inducing bend in DNA between the core promoter
and the binding site for the TF.
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From T. Brown, Genomes
Repressors of eukaryotic transcription
•Repression of initiation is very important in bacteria, but it
seemed unlikely that it plays a major control in eukaryotic
transcription.
•But, a growing number of DNA-binding proteins that repress
initiation were found.
•They bind at upstream promoter elements or at more distant
sites at silencers.
•Not really comprehensively studied in eukaryotes.
•Some repressors interact with GTFs (TFIIB, TFIID) – affect
assembly of preinitiation complex. They may act by inducing
chromatin formation over promoter, inducing silencing.
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From T. Brown, Genomes
Control over activators
Their activity has to be controlled
Activity can be regulated either by controlling the synthesis
or by controlling the activity to activate or repress transcription
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From T. Brown, Genomes
Control over the TFs
Direct activation – occurs when the extracellular signaling
compound is able to enter the cell
Indirect activation occurs when the extracellular signaling
compound in unable to cross the membrane and instead binds
to a cell surface receptor which transduces the signal to the
cell interior.
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Activators - stimulate or inhibit transcription by RNA
polymerase II
Two functional domains:
•DNA-binding domain
•Transcription activation domain
Also: may have dimerization domain (allows binding
of activators to each other)
DNA-binding domains
DNA binding domains have DNA binding motifs
1. Zinc-containing modules
• Zinc-fingers (in TFIIIA or Sp1)
• Zinc modules found in the glucocorticoid receptors
• Modules containing 2 zinc ions and 6 cysteins, found in
GAL4
2. Homeodomains – contain about 60 aa and resemble in
structure and function the helix-turn-helix DNA-binding
domains.HDs found in many activators, originally were
identified in homeobox proteins that regulate development of
Drosophila.
3. bZIP and bHLH motifs. The CCAAT/enhancer binding
protein, the MyoD protein and many eukaryotic TFs have a
highly basic DNA-binding motif linked to one or both of the
protein dimerization motifs known as leucine zippers and
helix-loop-helix (HLH) motifs.
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Transcription-activating domains
Most activators have one, some have more
Main classes:
1. Acidic domains. GAL4 –typical for this group. It has 49 aadomain with 11 acidic aa.
2. Glutamine-rich domains. The activator Sp1 has two such
domains, which are about 25% glutamine. One of these has
39 glutamines in a span of 143 aa. Also, Sp1 has two other
activating domains that do not fit into any of these main
categories.
3. Proline-rich domains. The activator CTF has a domain of
84 aa, 19 of which are prolines.
Structures of the DNA-binding motifs of activators
Zinc-fingers:
•Composed of anti-parallel β-strand, followed by an α-helix.
•The β-strand contains two cysteines, and the α-helix two histidines,
that are coordinated to a zinc ion.
•This coordination of aa to the metal ion helps to form the fingershaped structure.
•Specific recognition between the finger and its DNA target occurs
in the major groove.
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Structure of zinc
finger
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The GAL4 protein
GAL4 – yeast activator that controls set of genes responsible for
metabolism of galactose.
Each of these GAL4-responsible genes contains a GAL4 target site
upstream the transcription start site.These are upstream activator
sequences are called UAS.
GAL4 binds to UAS as a dimer.
•Its DNA-binding motif is located in the first 65aa of the protein.
•DNA binding motif of each monomer has 6 cysteines that
coordinate two Zn ions. The recognition module contains a short αhelix that protrudes into the DNA major groove and makes a specific
interaction there.
•Dimetization motif is found in residues 65-94.
Nuclear receptors
A third class of zinc module is found in nuclear receptors.
Nuclear receptors interact with variety of steroid and other
hormones.
They form hormone-receptor complexes that function as activators
by binding to enhancers , or hormone response elements.
Thus – they stimulate transcription of associated genes.
They have hormone-binding domain.
Some exist in cytoplasm complexed with heat-shock protein (hps
90). When hormones are present the receptors dissociate from hps,
bind hormone.
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Glucocorticoid receptor action
The effect of ligand binding on
the receptor is to convert it from
a repressing complex to
activating complex.
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Nuclear receptors
Other receptors are nuclear (TH receptor).
In the absence of hormone they bind to their respective enhancers
and repress transcription.
In the presence of hormones they form hormone-receptor
complexes in the nucleus and function as activators for the same
enhancers.
Environment determines whether the DNA element to which
protein binds, the thyroid response element serves as an enhancer
or as a silencer.
Homeodomains
Homeodomains are DNA-binding domains found in a large family
of activators. Their name comes from homeoboxes, that first were
discovered in regulatory genes in Drosophila, called homeotic
genes.
Mutations cause developmental abnormalities.
Homeodomains contain DNA-binding motif that functions the
same way as prokaryotic helix-turn-helix motifs in which a
recognition helix fits into the DNA major groove and makes
specific contacts there.
N-terminal arm nestles in the adjacent minor groove.
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The bZIP ans bHLH domains
bZIP and bHLH domains combine DNA binding and dimerization.
bZIP – leucine zipper
and bHLH - helix-loop-helix part
They are dimerization domains.
Leucine zipper
Two alpha helices, leucines
interact as zipper
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Crystal structure of the bZIP motif of GCN4 bound to its target.
The bZIP ans bHLH domains
Complex between the bHLH domain of MyoD and its DNA target.
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Independence of the domains of activators
DNA binding and transcription-activating domains of activator
proteins are independent modules.
We can make hybrid proteins with the DNA-binding domain of one
protein and the transcription-activation domain of the other one –
and the hybrid protein functions as an activator.
Functions of activators
Recruitment of preinitiation complexes.
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Interaction among activators
Dimerization – great advantage to tge activator, it increases the
affinity between activator and its DNA target.
Homodimers or heterodimers are formed.
The Jun-Fos dimer
Jun and fos are proto-oncogenes
Jun and Fos proteins are DNA-binding proteins of the bZIP family.
Jun dimers are one form of the activator AP1, but Jun and Fos
cooperate to bind DNA more tightly than Jun dimers (Fos dimers
do not bind at all).
Abundant form of AP1 is probably a Jun-Fos heterodimer.
bZIP domains are crucial for binding.
AP1 target TGACTCA.
Multiple enhancers
Many genes have more than one enhancer, they respond to multiple
stimuli.
Examples – metallothionine gene, codes for protein that help to
prevent metal poisoning.
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Architectural transcription factors
ATFs change the shape of DNA control region so that other
proteins can interact successfully to stimulate transcription.
Example – T-cell receptor alpha chain gene control region, which
contains three enhancers, binding sites for activators Ets-1, LEF-1
and CREB within just 112 bp of the transcription start site.
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Tom Maniatis observed similar phenomenon in the human
interpheron-β (IFN β) control region, which also has an enhancer
close to promoter.
Enhancer with its activators
Model of enhanceosome assembled at the human IFN β gene.
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Insulators – block activation of unrelated genes by nearby enhancers
– neutral barriers to the influence of neighboring elements
How do they work – not really known exactly.
Insulators – DNA elements that shield genes from activation or
repression by enhancers or silencers. They block such activation or
repression on one side of a DNA element, but not on the other.
Function: They establish boundaries between DNA regions in a
chromosome.
No model has been proposed yet as a model of insulator action that
can account for all the data available.
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Mediators
Model for activation of a CRE-linked gene.
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Regulation of transcription factors
Activators are subject to a variety of regulation mechanisms.
Some are regulated by
•Ligand binding
•Phosphorylation
•Binding to inhibitory proteins
•Proteilysis.
Signal transduction pathways begin with a signaling molecule that
interacts with a receptor on a cell surface, which send a signal inside
the cell and frequently leads to altered gene expression.
Many signal transduction pathways, including MAP kinase pathway,
rely on protein phosphorylation to pass the signal from one protein
from another.
This amplifies the signal at each step.
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A signal transduction pathway involving MAP kinase
References:
1. R. Weaver, Molecular Biology, 2005
2. B. Lewin, Genes VII, 2000
3. T. Brown, Genomes, 1999.
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