Initiation of transcription

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Chapter 20

• Initiation of transcription

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20.1 Introduction20.2 Eukaryotic RNA polymerases
consist of many subunits20.3 Promoter elements
are defined by mutations and footprinting20.4
RNA polymerase I has a bipartite promoter20.5
RNA polymerase III uses both downstream and
upstream promoters20.6 The startpoint for RNA
polymerase II20.7 TBP is a universal factor20.8
TBP binds DNA in an unusual way20.9 The basal
apparatus assembles at the promoter20.10
Initiation is followed by promoter
clearance20.11 A connection between
transcription and repair20.12 Promoters for RNA
polymerase II have short sequence elements20.13
Some promoter-binding proteins are
repressors20.14 Enhancers contain bidirectional
elements that assist initiation 20.15
Independent domains bind DNA and activate
transcription20.16 The two hybrid assay detects
protein-protein interactions20.17 Interaction of
upstream factors with the basal apparatus
3
Enhancer element is a cis-acting sequence that
increases the utilization of (some) eukaryotic
promoters, and can function in either orientation
and in any location (upstream or downstream)
relative to the promoter.
20.1 Introduction
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Figure 20.1 A typical gene transcribed by RNA
polymerase II has a promoter that extends
upstream from the site where transcription is
initiated. The promoter contains several short
(lt10 bp)sequence elements that bind transcription
factors, dispersed over gt200 bp. An enhancer
containing a more closely packed array of
elements that also bind transcription factors may
be located several kb distant. (DNA may be coiled
or otherwise rearranged so that transcription
factors at the promoter and at the enhancer
interact to form a large protein complex.)
20.1 Introduction
5
Amanitin (more fully a-amanitin)is a bicyclic
octapeptide derived from the poisonous mushroom
Amanita phalloides it inhibits transcription by
certain eukaryotic RNA polymerases, especially
RNA polymerase II.
20.2 Eukaryotic RNA polymerases consist of many
subunits
6
Figure 20.2 Eukaryotic RNA polymerase II has gt10
subunits.
20.2 Eukaryotic RNA polymerases consist of many
subunits
7
Cotransfection is the simultaneous transfection
of two markers.
20.3 Promoter elements are defined by mutations
and footprinting
8
Figure 20.3 Promoter boundaries can be determined
by making deletions that progressively remove
more material from one side. When one deletion
fails to prevent RNA synthesis but the next stops
transcription, the boundary of the promoter must
lie between them.
20.3 Promoter elements are defined by mutations
and footprinting
9
Figure 20.4 Transcription units for RNA
polymerase I have a core promoter separated by
70 bp from the upstream control element. UBF1
binds to both regions, after which SL1 can bind.
RNA polymerase I then binds to the core promoter.
The nature of the interaction between the factors
bound at the upstream control element and those
at the core promoter is not known.
20.4 RNA polymerase I has a bipartite promoter
10
Preinitiation complex in eukaryotic transcription
describes the assembly of transcription factors
at the promoter before RNA polymerase binds.
20.5 RNA polymerase III uses both downstream and
upstream promoters
11
Figure 20.5 Deletion analysis shows that the
promoter for 5S RNA genes is internal initiation
occurs a fixed distance (55 bp) upstream of the
promoter.
20.5 RNA polymerase III uses both downstream and
upstream promoters
12
Figure 20.6 Promoters for RNA polymerase III may
consist of bipartite sequences downstream of the
startpoint, with boxA separated from either boxC
or boxB. Or they may consist of separated
sequences upstream of the startpoint (Oct, PSE,
TATA).
20.5 RNA polymerase III uses both downstream and
upstream promoters
13
Figure 20.7 Initiation via the internal pol III
promoters involves the assembly factors TFIIIA
and TFIIIC, the initiation factor TFIIIB, and RNA
polymerase III.
20.5 RNA polymerase III uses both downstream and
upstream promoters
14
TATA box is a conserved AT-rich septamer found
about 25 bp before the startpoint of each
eukaryotic RNA polymerase II transcription unit
may be involved in positioning the enzyme for
correct initiation.
20.6 The startpoint for RNA polymerase II
15
Figure 20.8 RNA polymerases are positioned at all
promoters by a factor that contains TBP.
20.7 TBP is a universal factor
16
Figure 20.9 A view in cross-section shows that
TBP surrounds DNA from the side of the narrow
groove. TBP consists of two related (40
identical) conserved domains, which are shown in
light and dark blue. The N-terminal region varies
extensively and is shown in green. The two
strands of the DNA double helix are in light and
dark grey. Photograph kindly provided by Stephen
Burley.
20.7 TBP is a universal factor
17
Figure 20.10 The cocrystal structure of TBP with
DNA from -40 to the startpoint shows a bend at
the TATA box that widens the narrow groove where
TBP binds. Photograph provided by Stephen Burley.
20.7 TBP is a universal factor
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Figure 20.11 An initiation complex assembles at
promoters for RNA polymerase II by an ordered
sequence of association with transcription
factors.
20.8 The basal apparatus assembles at the promoter
19
Figure 20.12 Two views of the ternary complex of
TFIIB-TBP-DNA show that TFIIB binds along the
bent face of DNA. The two strands of DNA are
green and yellow, TBP is blue, and TFIIB is red
and purple. Photograph kindly provided by Stephen
Burley.
20.8 The basal apparatus assembles at the promoter
20
Figure 20.13 Phosphorylation of the CTD by the
kinase activity of TFIIH may be needed to release
RNA polymerase to start transcription.
20.8 The basal apparatus assembles at the promoter
21
Figure 20.14 Mfd recognizes a stalled RNA
polymerase and directs DNA repair to the damaged
template strand.
20.9 A connection between transcription and
repair
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Figure 14.28 The Uvr system operates in stages in
which UvrAB recognizes damage, UvrBC nicks the
DNA, and UvrD unwinds the marked region.
20.9 A connection between transcription and
repair
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Figure 20.15 The TFIIH core may associate with a
kinase at initiation and associate with a repair
complex when damaged DNA is encountered.
20.9 A connection between transcription and
repair
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Figure 14.37 A helicase unwinds DNA at a damaged
site, endonucleases cut on either side of the
lesion, and new DNA is synthesized to replace the
excised stretch.
20.9 A connection between transcription and
repair
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CAAT box is part of a conserved sequence located
upstream of the startpoints of eukaryotic
transcription units it is recognized by a large
group of transcription factors.
20.10 Promoters for RNA polymerase II have short
sequence elements
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Figure 20.16 Saturation mutagenesis of the
upstream region of the b-globin promoter
identifies three short regions (centered at -30,
-75, and -90) that are needed to initiate
transcription. These correspond to the TATA,
CAAT,
20.10 Promoters for RNA polymerase II have short
sequence elements
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Figure 20.17 Promoters contain different
combinations of TATA boxes, CAAT boxes, GC boxes,
and other elements.
20.10 Promoters for RNA polymerase II have short
sequence elements
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Table 20.17 Upstream transcription factors bind
to sequence elements that are common to mammalian
RNA polymerase II promoters.
20.10 Promoters for RNA polymerase II have short
sequence elements
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Table 20.17 Upstream transcription factors bind
to sequence elements that are common to mammalian
RNA polymerase II promoters.
20.10 Promoters for RNA polymerase II have short
sequence elements
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Figure 20.18 A transcription complex involves
recognition of several elements in the sea urchin
H2B promoter in testis. Binding of the CAAT
displacement factor in embryo prevents the
CAAT-binding factor from binding, so an active
complex cannot form.
20.10 Promoters for RNA polymerase II have short
sequence elements
31
Enhancer element is a cis-acting sequence that
increases the utilization of (some) eukaryotic
promoters, and can function in either orientation
and in any location (upstream or downstream)
relative to the promoter.
20.11 Enhancers contain bidirectional elements
that assist initiation
32
Figure 19.39 Indirect end-labeling identifies the
distance of a DNAase hypersensitive site from a
restriction cleavage site. The existence of a
particular cutting site for DNAase I generates a
discrete fragment, whose size indicates the
distance of the DNAase I hypersensitive site from
the restriction site.
20.11 Enhancers contain bidirectional elements
that assist initiation
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Figure 19.40 The SV40 minichromosome has a
nucleosome gap. Photograph kindly provided by
Moshe Yaniv.
20.11 Enhancers contain bidirectional elements
that assist initiation
34
Figure 20.19 An enhancer contains several
structural motifs. The histogram plots the effect
of all mutations that reduce enhancer function to
lt75 of wild type. Binding sites for proteins are
indicated below the histogram.
20.11 Enhancers contain bidirectional elements
that assist initiation
35
Figure 20.16 Saturation mutagenesis of the
upstream region of the b-globin promoter
identifies three short regions (centered at -30,
-75, and -90) that are needed to initiate
transcription. These correspond to the TATA,
CAAT,
20.11 Enhancers contain bidirectional elements
that assist initiation
36
Figure 20.20 An enhancer may function by bringing
proteins into the vicinity of the promoter. An
enhancer does not act on a promoter at the
opposite end of a long linear DNA, but becomes
effective when the DNA is joined into a circle by
a protein bridge. An enhancer and promoter on
separate circular DNAs do not interact, but can
interact when the two molecules are catenated.
20.11 Enhancers contain bidirectional elements
that assist initiation
37
Figure 20.21 DNA-binding and activating functions
in a transcription factor may comprise
independent domains of the protein.
20.12 Independent domains bind DNA and activate
transcription
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Figure 20.22 The GAL4 protein has independent
regions that bind DNA, activate transcription (2
regions), dimerize, and bind the regulator GAL80.
20.12 Independent domains bind DNA and activate
transcription
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Figure 20.23 The ability of GAL4 to activate
transcription is independent of its specificity
for binding DNA. When the GAL4 DNA-binding domain
is replaced by the LexA DNA-binding domain, the
hybrid protein can activate transcription when a
LexA operator is placed near a promoter.
20.12 Independent domains bind DNA and activate
transcription
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Figure 20.24 The activating domain of the tat
protein of HIV can stimulate initiation if it is
tethered in the vicinity by binding to the RNA
product of a previous round of transcription.
Activation is independent of the means
20.12 Independent domains bind DNA and activate
transcription
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Figure 20.25 The two hybrid technique tests the
ability of two proteins to interact by
incorporating them into hybrid proteins where one
has a DNA-binding domain and the other has a
transcription-activating domain.
20.12 Independent domains bind DNA and activate
transcription
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Figure 20.21 DNA-binding and activating functions
in a transcription factor may comprise
independent domains of the protein.
20.13 Interaction of upstream factors with the
basal apparatus
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Figure 20.26 An upstream transcription factor may
bind a coactivator that contacts the basal
apparatus.
20.13 Interaction of upstream factors with the
basal apparatus
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Figure 20.24 The activating domain of the tat
protein of HIV can stimulate initiation if it is
tethered in the vicinity by binding to the RNA
product of a previous round of transcription.
Activation is independent of the means
20.13 Interaction of upstream factors with the
basal apparatus
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Figure 20.11 An initiation complex assembles at
promoters for RNA polymerase II by an ordered
sequence of association with transcription
factors.
20.13 Interaction of upstream factors with the
basal apparatus
46
Figure 20.27 Upstream activators may work at
different stages of initiation, by contacting the
TAFs of TFIID or contacting TFIIB.
20.13 Interaction of upstream factors with the
basal apparatus
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1. Of the three eukaryotic RNA polymerases, RNA
polymerase I transcribes rDNA and accounts for
the majority of activity, RNA polymerase II
transcribes structural genes for mRNA and has the
greatest diversity of products, and RNA
polymerase III transcribes small RNAs. 2. None
of the three RNA polymerases recognize their
promoters directly. 3. The TATA box (if there is
one) near the startpoint, and the initiator
region immediately at the startpoint, are
responsible for selection of the exact startpoint
at promoters for RNA polymerase II.4. RNA
polymerase is found as part of much larger
complexes that contain factors that interact with
activators and repressors. 5. Promoters for RNA
polymerase II contain a variety of short
cis-acting elements, each of which is recognized
by a trans-acting factor. 6. Promoters may be
stimulated by enhancers, sequences that can act
at great distances and in either orientation on
either side of a gene.
Summary