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
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.)
20.1 Introduction
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.
20.2 Eukaryotic RNA polymerases consist of many subunits
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,
20.3 Promoter
elements are defined
by mutations and
footprinting
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
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.
20.5 RNA polymerase III uses both downstream and upstream promoters
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.
20.6 The startpoint for RNA polymerase II
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 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
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
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,
20.8 The basal apparatus
assembles at the promoter
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.
20.9 A connection
between
transcription and
repair
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 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
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
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
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
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
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 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
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.
20.11 Enhancers contain bidirectional elements
that assist initiation
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 transcription-activating
domain,
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,
20.13 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,
20.13 Interaction of upstream factors with the
basal apparatus
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