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Chapter
20
Initiation of
transcription
20.1 Introduction
20.2 Eukaryotic RNA polymerases consist of many subunits
20.3 Promoter elements are defined by mutations and footprinting
20.4 RNA polymerase I has a bipartite promoter
20.5 RNA polymerase III uses both downstream and upstream promoters
20.6 The startpoint for RNA polymerase II
20.7 TBP is a universal factor
20.8 TBP binds DNA in an unusual way
20.9 The basal apparatus assembles at the promoter
20.10 Initiation is followed by promoter clearance
20.11 A connection between transcription and repair
20.12 Promoters for RNA polymerase II have short sequence elements
20.13 Some promoter-binding proteins are repressors
20.14 Enhancers contain bidirectional elements that assist initiation
20.15 Independent domains bind DNA and activate transcription
20.16 The two hybrid assay detects protein-protein interactions
20.17 Interaction of upstream factors with the basal apparatus
20.1 Introduction
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
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
(<10 bp)sequence elements that bind transcription factors, dispersed over >200 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.)
20.2 Eukaryotic RNA polymerases consist
of many subunits
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
Figure 20.2
Eukaryotic
RNA
polymerase II
has >10
subunits.
20.3 Promoter elements are defined by
mutations and footprinting
Cotransfection is the
simultaneous
transfection of two
markers.
20.3 Promoter
elements are defined
by mutations and
footprinting
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.
20.4 RNA
polymerase I has a
bipartite promoter
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.
20.5 RNA polymerase III uses both downstream
and upstream promoters
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
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
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
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.
20.6 The startpoint for RNA polymerase II
TATA box is a conserved A·T-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.
20.7 TBP is a
universal factor
Figure 20.8
RNA
polymerases
are positioned
at all
promoters by a
factor that
contains TBP.
20.7 TBP is a
universal factor
Figure 20.9 A view in crosssection 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
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.
20.8 The basal apparatus
assembles at the promoter
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
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
Figure 20.13
Phosphorylation of
the CTD by the
kinase activity of
TFIIH may be
needed to release
RNA polymerase to
start transcription.
20.9 A connection between
transcription and repair
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
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
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
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.
20.10 Promoters for RNA polymerase II have
short sequence elements
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
Figure 20.16
Saturation
mutagenesis of
the upstream
region of the bglobin 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
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
Module
Consnesus
DNA bound
Factor
TATA box
TATAAAA
~10bp
TBP
CAAT box
GGCCAATCT
~22bp
CTF/NF1
GC box
GGGCGG
~20bp
SP1
Octamer
ATTTGCAT
~20bp
Oct-1
Octamer
ATTTGCAT
~23bp
Oct-2
kB
GGGACTTTCC
~10bp
NF kB
ATF
GTGACGT
~20bp
ATF
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
Module
Consnesus
DNA bound
Factor
TATA box
TATAAAA
~10bp
TBP
CAAT box
GGCCAATCT
~22bp
CTF/NF1
GC box
GGGCGG
~20bp
SP1
Octamer
ATTTGCAT
~20bp
Oct-1
Octamer
ATTTGCAT
~23bp
Oct-2
kB
GGGACTTTCC
~10bp
NF kB
ATF
GTGACGT
~20bp
ATF
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
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.
20.11 Enhancers contain bidirectional
elements that assist initiation
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
Figure 19.39 Indirect endlabeling 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
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
Figure 20.19 An
enhancer
contains several
structural motifs.
The histogram
plots the effect of
all mutations that
reduce enhancer
function to <75%
of wild type.
Binding sites for
proteins are
indicated below
the histogram.
20.11 Enhancers contain bidirectional elements
that assist initiation
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
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.
20.12 Independent domains bind DNA and
activate transcription
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
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
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
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
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 transcriptionactivating domain.
20.13 Interaction of upstream factors with the
basal apparatus
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
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
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
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
Figure 20.27
Upstream
activators may
work at different
stages of
initiation, by
contacting the
TAFs of TFIID
or contacting
TFIIB.
Summary
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.