Transcript RNA Polymerases

Section M—Transcription in eukaryotes
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M1 The three RNA polymerases:
characterization
and function
M2 RNA Pol Ⅰ genes: the ribisomal repeat
M3 RNA Pol Ⅲ genes: 5S and tRNA
transcription
M4 RNA Pol Ⅱ genes: promoters and enhancers
M5 General transcription factors and RNA Pol
Ⅱ
initiation
M1 The three RNA polymerases:
characterization and function
Transcription and its control are much more complex in eukaryotes.
There are 3 RNA polymerases, each specific for a subset of RNAs.
Eukaryotic RNA polymerases
The mechanism of eukaryotic transcription is similar to that in prokaryotes. However,
the large number of polypeptides associated with the eukaryotic transcription
machinery makes it far more complex. Three different RNA polymerase complexes
are responsible for the transcription of different types of eukaryotic genes. The
different RNA polymerases were identified by chromatographic purification of the
enzymes and elution at different salt concentrations(Topic B4). Each RNA polymerase
has a different sensitivity to the fungal toxin α-amanitin and this can be used to
distinguish their activities.
●RNA polynerase Ⅰ(RNA pol Ⅰ) transcribes most rRNA genes. It is located in the
nucleoli and is insensitive to α-amanitin.
●RNA polynerase Ⅱ(RNA pol Ⅱ) transcribes all protein-coding genes and some
small nuclear RNA(snRNA) genes. It is located in the nucleoplasm and is very
sensitive to α-amanitin.
●RNA polynerase Ⅲ(RNA pol Ⅲ) transcribes the genes for tRNA, 5S rRNA, U6
snRNA and certain other small RNAs. It is located in the nucleoplasm and is
moderately sensitive to α-amanitin.
In addition to these nuclear enzymes, eukaryotic cells contain additional polymerase
in mitochondria and chloroplasts.
There Are Many
Functional Classes of
RNA
mRNA
rRNA
tRNA
snRNA
scRNA
7S RNA
micro RNA
RNA polymerase subunits
– 500-700kDa, 12+ subunits, most of RNA pol II structures are
determined. The genes encoding the two largest subunits of each
RNA polymerase have homology to each other.
• The largest subunits of each eukaryotic RNA polymerase is similar
to the β’ subunit of the E. coli polymerase, and the second largest
subunit is similar to the βsubunit which contains the actiove site of
the E. coli enzyme.
• Two subunits which are common to RNA Pol I and RNA PolIII, and a
further subunit which is specific to RNA Pol II, have homology to the
E. coli RNA polymerase αsubunit.
• At least five other smaller subunits are common to the three
different polymerases. Each polymerase also contains an additional
four to seven subunits which are only present in one type.
Similar Structures of Bacterial (left) and
Eukaryotic (right) RNA Polymerases
Eukaryotic RNA polymerase activities
Like bacterial RNA polymerases, each of the eukaryotic enzymes
catalyzes transcription in a 5’ to 3’ direction and synthesizes RNA
complementary to the antisense template strand.
•The reaction requires the precursor nuckeotides ATP，GTP，
CTP and UTP and does not requires a primer for transcription
initiation.
•The purified eukaryotic RNA polymerases, unlike the purified
bacterial enzymes, require the presence of additional initiation
proteins before they are able to bind to promoters and initiate
transcription.
The CTD of RNA Pol II
CTD---C-terminal domain
RNA Pol II RPB1 subunit has (CTD) with repeat
(YSPTSPS)n, n=26-52,
In vitro studies have shown that the CTD sequence may be
phosphorylated at the serines and tyrosines.
Phosphorylate /Unphosphorylated
Unphosphorylated to initiate transcription
Phosphorylated for elongation
M2 RNA PolⅠ genes: the ribosomal repeat
RNA polymerase Ⅰ( RNA pol Ⅰ) is responsible for the
continuous synthesis of rRNA during interphase. Human
cells contain five clusters of around 40 copie of rRNA gene
situated on different chromosomes (see Fig.1 and Topic D4).
Each rRNA gene produces a 45S rRNA transcript which is
about 13000 nt long(see the Topic D4).
This transcript is cleaved to give one copy each of the 28S
RNA (5000 nt), 18S(2000nt) and 5.8S (160 nt) rRNA (see
Topic O1).
The continuous transcription of multiple gene copies of the
RNAs is essential for sufficient production of the processed
rRNAs which are packaged into ribosomes.
Ribosomal RNA transcription units
Promoter
18S
5.8S
28S
Transcription
45S transcript
18S
5
18S
rRNA
5.8S
28S
3
Cleavage
(the light pink regions
are degraded)
5.8S
rRNA
28S
rRNA
Role of the nucleolus
Each rRNA cluster is known as a nucleolar organizer region, since the
nucleolus contains large loops of DNA correspondind to the gene
clusters. After a cell emerges from mitosis, rRNA synthesis restarts and
tiny nucleoli appear at the chromosomal locations of the rRNA genes.
During active rRNA synthesis, the pre-rRNA transcripts are packed
along the rRNA genes and may be visualized in the electron microscope
as ‘Chrismas tree structures’.
In these structures, the RNA transcripts are densely packed along the
DNA and stick out perpendicularly from the DNA.
Short transcripts can be seen at the start of the gene, which get longer
until the end of the transcription unit, which is indicated by
disappearance of the RNA transcripts.
RNA Pol I promoters
Mammalian pre-rRNA gene promoters have a bipartite
transcription control region(Fig.2). The core element includes
the transcription start site and encompasses bases -31 to +6.
This sequence is essential for transcription.
An additional element of around 50-80 bp named the upstream
control element(UCE) begins about 100 bp upstream from the
start site (-100).
The UCE is responsible for an increase in transcription of
around 10- to 100-fold compared with that from the core
element alone.
Upstream binding factor
UBF：A specific DNA-binding
protein,called upstream binding
factor, binds to the UCE
SL1: An additional factor called
selectivity factor 1, is essential for
RNA Pol Ⅰtranscription. SL1 binds
to and stabilizes the UBF-DNA
complex and interacts with free
downstream part of the core
element.
TBP：One of the subunits of SL1,
called TATA-binding protein, is
required for initiation by all three
eukaryotic RNA polymerases.
TAFI s（TBP-associated
factors）：as the other subunits
of SL1 and required for RNA
polyⅠtranscription called TAFⅠs。
Schematic model for rRNA transcription initiation
M3 RNA Pol ⅢGenes: 5S and tRNA
transcription
RNA polymerase Ⅲ(RNA poly Ⅲ) is a complex of at
16 defferent subunits. Like RNA Pol Ⅱ, it is located
in the nucleoplasm.
RNA polymerase Ⅲ synthesizes the precursors of 5S
rRNA, the tRNAs and snRNA and cytosolic RNAs.
tRNA gene transcription
Why are the highly conserved
sequences within the tRNA also
highly conserved promoter DNA
sequences?
A box：5’-TGGCNNAGTGG-3’
B box：5’-GGTTCGANNCC-3’
TFIIIB and TFIIIC are required for
tRNA gene transcription.
• TFIIIB allows RNA Poly Ⅲ to bind
and initiate transcription.
• TFIIIC is an assembly factor for
the positioning of the initiation
factor TFⅢB.
Initiation of transcription at a eukaryotic tRNA promoter
5S rRNA gene transcription
The promoters of the 5S
rRNA genes contain C box
and A box as internal
control regions.
5S rRNA transcription
initiation needs an additional
assembly factor TFⅢA
relative to the tRNA
transcription initiation.
TFⅢA acts to bind to C box
and stabilize the interaction
between TFⅢC and 5S
rRNA.
Initiation of transcription at a eukaryotic 5S rRNA promoter
Alternative RNA Pol Ⅲ promoters and
RNA Pol Ⅲ termination
Many RNA Pol III genes also rely on upstream sequences for the
regulation of their transcription. Some promoters such as the U6
small nuclear RNA (U6 snRNA ) and small RNA genes from the
Epstein-Barr virus use only regulatory sequences upstream from
their transcription start sites.
The coding region of the U6 snRNA has a characteristic A box.
However, this sequence is not required for transcription. The U6
snRNA upstream sequence contains sequence typical of RNA Pol II
promoters, including a TATA box at bases -30 to -23. these
promoters also share several other upstream transcription factor
binding sequences with many U RNA genes which are transcribed
by RNA Pol II. These observations suggest that common
transcription factors can regulate both RNA Pol II and RNA Pol
III genes.
RNA Pol Ⅲ termination
Termination of transcription by RNA Pol Ⅲ appears
only to require polymerase recognition of a simple
nucleotide sequence consisting of dA residues, whose
termination efficiency is affected by surrounding
sequence.
Thus the sequence 5’-GCAAAAGC-3’ is an efficient
termination signal in the Xenopus borealis somatic
5SrRNA gene.
M4 RNA Pol II genes: promoters and
enhancers
RNA Polymerase II (RNA Pol II) is located in the
nucleoplasm. It is responsible for the transcription of all
protein-coding genes and some small nuclear RNA genes.
The pre-mRNAs must be processed after synthesis by cap
formation at the 5’-end of the RNA and poly (A) addition
at the 3’-end, as well as removal of introns by splicing.
Promoters
Transcriptional
start site
TATA box
Coding-strand sequences: TATAAAA Py2CAPy5
–100
–50
–25 +1
GC + CAAT boxes
DNA
Transcription
TATA box: Many eukaryotic promoters contain a sequence called the
TATA box around 25-35 bp upstream from the start site of transcription.
It has the 7 bp consensus sequence 5’-TATA（A/T）A（A/T）-3’
although it is now known that the protein which binds to the TATA box,
TBP, binds to an 8 bp sequence that includes an additional downstream
base pair, whose identity is not important.
Initiator element: The initiator element is located around the
transcription strt site. Many initiator elements have a C at -1 and A at +1.
Upstream regulatory elements
These elements are found in many genes which vary widely in
their levels of expression in different tissues.
Two common examples are SP1 box, which is found upstream
of many genes both with and without TATA boxes, and the
CCAAT box.
Promoters may have one, both or multiple copies of these
sequences.
These sequences which are often located within 100-200 bp
upstream from the promoter are referred to as upstream
regultory elements (UREs) and play an important role in
ensuring efficient transcription from the promoter.
Enhancers
Transcription from many eukaryotic promoters can be stimulated by
control elements that are located many thousands of base pairs away
from the transcription start site. This kind of elements are called as
enhancer. Classically, enhancers have the following general
chracteristics:
• They exert strong activation of transcription of a ;linked gene from the
correct start site.
•They activate transcription when placed in either orientation with respect to
linked genes.
•They are able to function over long distances of more than 1 kb whether
from an upstream or downstream position relative to the start site.
•They exert preferential stimulation of the closest of two tandem promoters.
M5 General transcription factors and RNA
Pol II initiation
RNA Pol II basal transcription fators：A serial of nuclear
transcription factors have been identified, purified amd cloned. These
are required for basal trancription initiation from RNA Pol II promoter
sequences in vitro and named as TFIIA，TFIIB，TFIIC，TFIID.
They have been shown to assemble on basal promoters in a specific
order and they may be subject to multiple levels of regulation.
TFIID：Inn promoters containing a TATA box, the RNA Pol II
transcription factor TFIID is responsible for binding to this key promoter
element. The binding of TFIID to the TATA box is the earliest stage in the
formation of the RNA Pol II transcription initiation complex. It seems
that in mammalian cells, TBP binds to the TATA box and is then joined by
at least eight 由TBP和TAFIIs to form TFIID.
TBP
TBP is present in all three enkaryotic
transcription complexes and clearly plays a
major role in transcription initiation. TBP is a
monomeric protein, with a highly conserved Cterminal domains of 180 residues and this
conserved domain functions as well as the fulllength protein in in vivo transcription.
TBP structure
TBP has been shown to have
saddle structure with an overall
dyad symmetry, but two halves
of the molecule are not identical.
TBP interacts with DNA in the
minor groove so that the inside
of the saddle binds to DNA at the
TATA box and the outside
surface of the protein is available
for interactions with other
protein factors. Binding of TBP
deforms the DNA so that it is
bent into the inside of the saddle
unwound. This results in a kink
of about 45° between the first
two and last two base pariss of
the 8 bp TATA element.
TFIIA, TFIIB and RNA polymearse binding
TFIIA: TFIIA binds to TFIID and enhances TFIID
binding to the TATA box, stabilizing the TFIID-DNA
complex. TFIIA is made up of at least three subunits.
TFIIB and RNA polymearse binding: Once TFIID has
bound to the DNA, another transcription factor, TFIIB,
binds to TFIID. TFIIB can also bind to the RNA
polymerase. This seems to be an important step in
transcription initiation since TFIIB asts as a bridging fator
allowing recruitment of the polymerase to the complex
togather with a further fator, TFIIF.
Factors binding after RNA polymerase
After RNA polymerase binding, three other transcription
factors, TFIIE, TFIIH, and TFIIJ, rapidly asociate with
the compex.
These proteins are necessary for transcription in vitro and
associate with the complex in a defined order.
TFIIH is a large compex which is made up of at least five
subunits. TFIIJ remains to fully characterized.
CTD phosphorylation by TFIIH
TFIIH is a large multicomponent protein compex which
contains both kinase and helicase activity.
Activation of TFIIH results in phophorylation of the
carboxyl-terminal domain (CTD) of the RNA polymerase.
This phosphorylation results on formation of a
processive RNA polymerase complex and allows the RNA
polymerase to leave the promoter region.
TFIIH therefore seems to have a very important function
in control of transcriptiom elongation.
TFIIA binds to TFIID and
enhances TFIID binding to
TATA box.
TFIID
TATA box
TFIIB binds to TFIID.
TFIID
TFIIA TFIIB
TFIIB acts as a bridge to bind to
Once TFIID has
RNA polymerase II/TFIIF.
bound to the DNA，
TFIIB binds to TFIID.
TFIID
TFIIA
TFIIF
RNA polymerase
TFIIE and TFIIH bind
to RNA polymerase II.
Transcription factors
After RNA polymerase
binding, three other
transcription factors,
TFIIE, TFIIH, and TFIIJ,
rapidly asociate with
the compex
Activation of TFIIH results in
phophorylation of the
carboxyl-terminal domain
(CTD) of the RNA
polymerase. This
phosphorylation results on
formation of a processive
RNA polymerase complex
and allows the RNA
polymerase to leave the
promoter region.
The initiator transcription complex
Many RNA Pol II promoters which do not contain a
TATA box have an initiator element overlapping their
start site.
It seems that at these promoters TBP is recruited to the
promoter by a further DNA-binding protein which binds
to the initiator element.
TBP the recruits the other transcription factors and RNA
polymerase in a manner similar to that which occurs in
TATA box promoters.
RNA pol. Speed
Vmax = 50 nts / sec
About 1/10th DNA polymerase
Which needs to be fast:
Initiated at very few points
Only ~ 10 DNA pol molecules / cell
Each has to be very fast
~3,000 molecules / cell
Transcription simultaneously at many points
RNA pol. Fidelity
Error rate of ~ 10-4 to 10-5.
Much greater than DNA pol
Less than expected just from W.-C base-pairing
Suggests proof-reading
Details of proof-reading not understood.
Homework
What about the dsDNA sequence tells RNA
polymerase where to start?
Be sure to be able to distinguish between
prokaryotes and eukaryotes in your answer.