A1258150538AiqCM - PowerPoint PPT Presentation

Title: A1258150538AiqCM


1
Molecular Biology
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.
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Molecular Biology
Promoters
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.
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Molecular Biology
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.
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Molecular Biology
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.

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Molecular Biology
M5 General transcription factors and RNA Pol II
initiation
RNA Pol II basal transcription fatorsA 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. TFIIDInn 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 ?TBP?TAFIIs to form TFIID.
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Molecular Biology
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
C-terminal domains of 180 residues and this
conserved domain functions as well as the
full-length protein in in vivo transcription.
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Molecular Biology
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.
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Molecular Biology
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.
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Molecular Biology
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.
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Molecular Biology
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.
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Molecular Biology
TFIIA binds to TFIID and enhances TFIID binding
to TATA box.
Once TFIID has bound to the DNA,TFIIB binds to
TFIID.
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Molecular Biology
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.
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Molecular Biology
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.
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Molecular Biology
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
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Molecular Biology
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.
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Molecular Biology
Your 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.