Section M

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Section M Transcription in eukaryotes
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Contents

• M1 The three RNA polymerases characterization
  and function
• Eukaryotic RNA polymerase, RNA polymerase
  subunits, Eukaryotic RNA polymerase activities,
  The CTD of RNA Pol?
• M2 RNA Pol ? genes the ribosomal repeat
• Ribosomal RNA genes, Role of the nucleolus,
  RNA Pol? promoters, Upstream binding factor,
  Selectivity factor 1, TBP and TAF1s, Other rRNA
  genes
• M3 RNA Pol ? genes 5s and tRNA transcription
• RNA polymerase ?, tRNA genes, 5s rRNA genes,
  Alternative RNA Pol? promoters, RNA Pol?
  termination
• M4 RNA Pol ? genes promoters and enhancers
• RNA polymerase ?, Promoters, Upstream
  regulatory elements, Enhancers
• M5 General transcriptiion factors and RNA Pol ?
  initiation
• RNA Pol? basal transcription factors, TF?D,
  TBP, TF?A, TF?B and RNA polymerase binding,
  Factors binding after RNA polymerase, CTD
  phosphorylation by TF?H, The initiator
  transcriptiom complex

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M1 The three RNA polymerases characterization
and function Eukaryotic RNA polymerase

• Three eukaryotic polymerases transcribe different
  set of genes. Their activities are distinguished
  by their sensitivities to the fungal toxin(??)
  a-amanitin (a-????).
• RNA polymerase I is located in the nucleoli. Its
  responsible for the synthesis of the precursors
  of most rRNAs.
• RNA polymerase II is located in the nucleoplaam
  and is responsible for the synthesis mRNA
  precursors and some small nuclear RNAs.
• RNA polymerase III is located in the
  nucleoplasm.Its responsible foe the synthesis of
  the precursors of 5s rRNA, tRNAs and other small
  nuclear and cytosolic(?????) RNAs.

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Three eukaryotic polymerases
Type Location Substrate a-amanitin
RNA Pol I Nucleoli Most rRNAs gene Insensitive
RNA Pol II Nucleo-plasm All protein-coding genes and some snRNA genes Very sensitive
RNA Pol III Nucleo-plasm tRNAs, 5S rRNA, U6 snRNA and other small RNAs Moderately sensitive
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M1 The three RNA polymerases characterization
and function RNA polymerase subunits

• Each eukaryotic polymerase contains 12 or more
  subunits.
• the two largest subunits are similar to each
  other and to the b and b subunits of E. coli
  RNA Pol.

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M1 The three RNA polymerases characterization
and function Eukaryotic RNA polymerase
activities

• Transcription mechanism is similar to that of E.
  coli polymerase (How?)
• Different from bacterial polymerasae, they
  require accessory factors for DNA binding.

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M1 The three RNA polymerases characterization
and function The CTD of RNA Pol?

• The C-terminus of RNA Pol II contains a stretch
  of seven amino acids that is repeated 52 times in
  mouse enzyme and 26 times in yeast.
• The heptapeptide sequenc is Tyr-Ser-Pro-Thr-Ser-P
  ro-Ser
• This repeated sequence is known as carboxyl
  terminal domain (CTD)
• The CTD sequence may be phosphorylated at the
  serines and some tyrosines

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• 5. The CTD is unphosphorylated at transcription
  initiation, and phosphorylation occurs during
  transcription elongation as the RNA Pol II leaves
  the promoter (In vitro results).
• 6. Because it transcribes all eukaryotic
  protein-coding gene, RNA Pol II is the most
  important RNA polymerase for the study of
  differential gene expression. The CTD is an
  important target for differential activation of
  transcription elongation.

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M2 RNA Pol ? genes the ribosomal repeat
Ribosomal RNA genes

• A copy of 18S, 5.8S and 28S rRNA genes is
  organized as a single transcription unit in
  eukayotes. A 45S rRNA transcript (13 000 nt
  long) is produced during transcription, which is
  then processed into 18S, 5.8S and 28S rRNA.
• Pre-rRNA transcription units are arranged in
  clusters in the genome as long tandem arrays
  separated by nontranscribed spacer squences.

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1. Continuous transcription of multiple copies of
   rRNA genes by RNA Pol I is essential to produce
   sufficient rRNAs which are packaged into
   ribosomes.
2. The arrays of rRNA genes (rRNA cluster) loop
   together to form the nucleolus and are known as
   nucleolar (???)organizer regions.
3. During active rRNA synthesis, the pre-rRNA
   transcripts are packaged along the rRNA genes,
   visualizing in the electronic microscope as
   Christmas tree structures.

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M2 RNA Pol ? genes the ribosomal repeat
Role of the nucleolus

• Pre-rRNA is synthesized by RNA polymerase I (RNA
  Pol I) in the nucleolus.
• The arrays of rRNA gene loop together to form the
  nucleolus and are know as nucleolar organizer
  regions.

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We should clearly understand what a promoter is
before further study ? Lets review the concept.
A promoter is the site on the DNA to which an RNA
polymerase molecule binds prior to initiating
transcription.
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Antisense or template DNA strand the strand
from which it copies. Sense or coding strand
the other strand, to which it is identical RNA.

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The nucleotide 1 in transcription The
nucleotide at which transcription is initiated as
1. The nucleotide -1 in transcription The
nucleotide that precedes nucleotide 1 is
initiated as -1.
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Upstream Those portions of the DNA preceding the
initiation site(or start point)(toward the 3 end
of the template). Downstream Those portions of
the DNA succeeding the initiation site(or start
point)(toward the 5 end of the template).
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Prokaryotic promoter sequences
Be absolutely essential to start transcription in
prokaryotes.
-10 position ( called Pribnow box,TATAAT) -35
position (TTGACA)
Allows a very high transcription rate.
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Then ,we can know that the bacterial promoter
almost always contains some version of the
following elements
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Eukaryotic promoters
Eukaryotic promoters are extremely diverse and
are difficult to characterize. They typically
lie upstream of the gene and can have regulatory
elements several kilobases away from the
transcriptional start site.
(Fig.11.19)
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Many eukaryotic promoters, but by no means all,
contain a core promoter element, a TATA box
(sequence 5-TATAAA-3), which in turn binds a
TATA binding protein which assists in the
formation of the RNA polymerase transcriptional
complex.
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The TATA box typically lies very close to the
transcriptional start site (often within 50
bases). Eukaryotic promoter regulatory sequences
typically bind proteins called general
transcription factors(GTFs) which are involved in
the formation of the transcriptional complex.
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M2 RNA Pol ? genes the ribosomal repeat
RNA Pol? promoters

• Generally consists of a bipartite sequence in the
  region preceding the start site, including core
  element and the upstream control elements (UCE).
• RNA Pol I promoters in human cells are best
  characterized.
• Core element -45 to 20, sufficient for
  transcription initiatiation.
• UCE -180 to -107, to increase the transcription
  efficiency.
• Both regions are rich in GC, with 85 identity.
  

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M2 RNA Pol ? genes the ribosomal repeat
Upstream binding factor

• A specific DNA-binding protein that binds to UCE,
  as well as a different site in the upstream of
  the core element, causing the DNA to loop between
  the two sites. (two binding sites have no obvious
  similarity)
• UBF is essential for high level of transcription,
  and low level of expression occurs in its absence

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M2 RNA Pol ? genes the ribosomal repeat
Selectivity factor 1

• Does not bind to promoters by itself
• Binds to and stabilizes the UBF-DNA complex.
• Interacts with the free downstream part of the
  core element.
• Recruit RNA Pol I to bind and to initiate the
  transcription.

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M2 RNA Pol ? genes the ribosomal repeat
TBP and TAF1s

• SL1 consists of 4 proteins.
• TBP (TATA-binding protein) a factor also
  required for initiation by RNA Pol II and III. A
  critical general factor in eukaryotic
  transcription that ensures RNA Pol to be properly
  localized at the startpoint.
• Other three subunits are referred to as
  TBP-associated factors (TAFIs) that are specific
  for RNA Pol I transcription.

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• The initiation complex assembles in three stages

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M2 RNA Pol ? genes the ribosomal repeat
Other rRNA genes

• In a simple eukaryote, Acanthamoeba, the rRNA
  genes have only one control element (promoter)
  around 12-72 bp upstream from the transcription
  start site.
• Simple initiation
• TIF (homolog of SL-1) binds to the promoter ? RNA
  Pol I bind ? TIF remains bound and the RNA Pol I
  is released for elongation.

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M3 RNA Pol ? genes 5s and tRNA transcription
RNA polymerase ?

• 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).

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M3 RNA Pol ? genes 5s and tRNA transcription
tRNA genes

• The initial transcripts of tRNA genes need to be
  processed to produce the mature tRNA.
• The transcription control regions of tRNA lies
  after the start site within the transcribed
  region. The two highly conserved control
  sequences are called A box (5-TGGCNNAGTGG) and B
  box (5-GGTTCGANNCC).

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• A box and B box also encode important sequences
  in the tRNA itself, the D-loop and T?C-loop.
• Therefore, the highly conserved sequence in tRNAs
  are also highly conserved promoter DNA sequences.
  
• 3. Two complex DNA-binding factors required for
  tRNA transcription initiation
• TFIIIC---binds to both the A and B boxes, an
  assembly factor for positioning TFIIIB.

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TFIIIB (1) binds 50 bp upstream from the A box,
but has no sequence specificity and the binding
position is determined by the DNA bound TFIIIC.
(2) consists of three subunits, one of which is
TBP, the general initiation factor the second is
called BRF (TFIIB-related factor) and the third
is called B.
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TFIIIC A and B boxes binding and a assembly
factor to position TFIIIB
TFIIIB DNA binding and RNA Pol III recruiting
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M3 RNA Pol ? genes 5s and tRNA transcription
5s rRNA genes

• Tandemly arranged in a gene cluster. (In human,
  there is a single cluster of around 2000 genes.)
• Transcription control regions (promoters) are
  organized similar to those of tRNA, except that C
  box is in place of B box. C box 81-99 bp A
  box 50-65

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3. Transcription factors (1) The C box acts
as the binding site for TFIIIA. (2) TFIIIA
acts as an assembly factor which allows TFIIIC to
interact with the 5S rRNA promoter. (3) The
A box may also stabilize TFIIIC binding. (4)
TFIIIC is then bound to DNA site near 1.
(5) TFIIIB and TFIIIC interact to recruit RNA Pol
III to initiate transcription.
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M3 RNA Pol ? genes 5s and tRNA transcription
Alternative RNA Pol? promoters

• Many RNA Pol III genes also rely on upstream
  sequences for regulation of their transcription
• e.g. U6 snRNA and Epstein-Barr virus
• Use only regulatory genes upstream from their
  transcription start sites.

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• U6 snRNA
• The coding region contains a characteristic A box
  that is not required for transcription.
• The upstream sequence contains sequences typical
  of RNA Pol II promoters, including a TATA box at
  bases 30 to 23.
• Shares several other transcription factor binding
  sequences with many U RNA genes which are
  transcribed by RNA Pol II
• Suggestion common transcription factors can
  regulate both RNA Pol II and Pol III genes

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M3 RNA Pol ? genes 5s and tRNA transcription
RNA Pol? termination

• The RNA polymerase can terminate transcription
  without accessory factors. A cluster of A
  residue is often sufficient for termination.
  Xenopus borealis terminator 5-GCAAAAGC-3

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M4 RNA Pol ? genes promoters and enhancers
RNA polymerase ?

• located in nucleoplasm
• catalyzing the synthesis of the mRNA precursors
  for all protein-coding genes.
• RNA Pol ?-transcribed pre-mRNAs are processed
  through cap addition, poly(A) tail addition and
  splicing.

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M4 RNA Pol ? genes promoters and enhancers
Promoters

• Most promoters contain a sequence called the TATA
  box around 25-35 bp upstream from the start site
  of transcription. It has a 7 bp consensus
  sequence 5-TATA(A/T)A(A/T)-3.
• TBP binds to TATA box that includes an additional
  downstream bp.

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• TATA box acts in a similar way to an E. coli
  promoter 10 sequence to position the RNA Pol II
  for correct transcription initiation. The spacing
  but not the sequence between the TATA box and the
  start site is important. Transcription starts
  with an adenine 50 of the time.

Some eukaryotic genes contain an initiator
element instead of a TATA box. The initiator
element is located around the transcription start
site. Other genes have neither a TATA box nor an
initiator element, and usually are transcribed at
very low rates.
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M4 RNA Pol ? genes promoters and enhancers
Upstream regulatory elements

• The basal elements (the TATA box and initiator
  elements) primarily determine the location of
  the startpoint, and sponsor initiation only at a
  rather low level.
• Upstream regulatory elements (URE) such as the
  SP1 box and CCAAT boxes, greatly increase the
  frequency of initiation. URE is located within
  100-200 bp from the promoter, and plays an
  important role in ensuring efficient
  transcription.

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M4 RNA Pol ? genes promoters and enhancers
Enhancers

• Enhancers
• Sequence elements which can activate
  transcription from thousands of base pairs
  upstream or downstream.

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• General characteristics of Enhancers
• Exert strong activation of transcription of a
  linked gene from the correct start site.
• activate transcription when placed in either
  orientation with respect to linked genes
• Able to function over long distances of more than
  1 kb whether from an upstream or downstream
  position relative to the start site.
• Exert preferential stimulation of the closets of
  two tandem promoters

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M5 General transcriptiion factors and RNA Pol ?
initiation RNA Pol? basal
transcription factors

• A complex series of basal transcription factors
  have been characterzed which bind to RNA Pol II
  promoters and together initiate transcription.
• These factors and their component subunits are
  still being identified.
• They were originally named TFIIA, TFIIB TFIIC.

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M5 General transcriptiion factors and RNA Pol ?
initiation
TF?D

• Multiprotein Complex, including TBP, other
  proteins are known as TAFIIs.
• TBP is the only protein binds to TATA box

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M5 General transcriptiion factors and RNA Pol ?
initiation
TBP

• 1. a general transcription factor bound to DNA at
  the TATA box.
• 2. a general transcription required by all 3 RNA
  pol.
• 3. Has a saddle structure with an overall dyad
  symmetry.

Outer surface (with ?)
TBP
DNA
Inner surface (with ?)
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M5 General transcriptiion factors and RNA Pol ?
initiation
TF?A

• TFIIA
• binds to TFIID
• stabilizes TFIID-DNA complex
• contains at least 3 subunits

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M5 General transcriptiion factors and RNA Pol ?
initiation TF?B and RNA polymerase
binding

• TFIIB RNA Pol binding
• binds to TFIID
• Binds to RNA Pol with TFIIF

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M5 General transcriptiion factors and RNA Pol ?
initiation Factors binding after RNA
polymerase

• After RNA polymerase binding, TFIIE, TFIIH and
  TFIIJ associate with the transcription complex in
  a defined binding sequence.
• Each of these proteins is required for
  transcription in vitro.

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• 4-1 TFIIE binding
• Necessary for transcription

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• 4-2 TFIIJ, TFIIH binding
• Necessary for transcription

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5. phosphorylation of the polymerase CTD by
TFIIH Formation of a processive RNA polymerase
complex and allows the RNA Pol to leave the
promoter region.
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M5 General transcriptiion factors and RNA Pol ?
initiation CTD phosphorylation by
TF?H

• TFIIH phosphorylates(????) the carboxy-terminal
  domain (CTD,???????) of RNA Pol II.
• This results in formation of a processive
  polymerase complex.

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M5 General transcriptiion factors and RNA Pol ?
initiation The initiator transcriptiom
complex

• For TATA-box lacking RNA Pol II promoters, TBP is
  recruited to the initiator element 0verlapping
  the start site by some DNA-binding proteins, TBP
  then recruit the other transcription factors and
  polymerase similar to TATA box gene
  transcription.

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Multiple choice questions

• 1. Which one of the following statements about
  eukaryotic RNA polymerases I, II and III is
  false?
• A RNA Pol II is very sensitive toa-amanitin.
• B RNA Pol II is located in th nucleoplasm.
• C RNA Pol III transcribes th genes for tRNA.
• D eukaryotic cells contain other RNA
  polymerases in addition to RNA Pol I, RNA Pol II
  and RNA Pol III.
• E each RNA polymerase contains subunits with
  homology to subunits of the E. coli RNA
  polymerase as well as additional subunits, which
  are unique to each polymerase.
• F the carboxyl end of RNA Pol II contains a
  short sequence of only seven amino acids which is
  called the carboxyl-terminal domain (CTD) and
  which may be phosphorylated.
• 2. Which two of the following statements about
  RNA Pol I genes are true?
• A RNA Pol I transcribes the genes for ribosomal
  RNAs.
• B human cells contain 40 clusters of five
  copies of the rRNA gene.
• C the 185, 5.85 and 285 rRNAs are synthesized
  as separate transcripts.
• D RNA Pol I transcription occurs in the
  nucleoplasm.
• E RNA Pol I transcription occurs in the
  cytoplasm.
• F rRNA gene clusters are known as nucleolar
  organizer regions.

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• 3. Which one of the following statements about
  RNA Pol I transcription is false?
• A in RNA Pol I promoters the core element is
  1000 bases downstream from the upstream control
  element (UCE).
• B upstream binding factor (UBF) binds to both
  the UCE and the upstream part of the core element
  of the RNA Pol I promoter.
• C selectivity factor SLl stabilizes the UBF-DNA
  complex.
• D SL1 contains several subunits including the
  TATA-binding protein TBP.
• E in Acanthamoeba there is a single control
  element in rRNA gene promoters.
• 4. Which two of the following statements about
  RNA Pol III genes are true?
• A the transcriptional control regions of tRNA
  genes lie upstream of the start of transcription.
  
• B highly conserved sequences in tRNA gene coding
  regions are also promoter sequences.
• C TFIIIC contains TBP as one of its subunits.
• D TFIIIB is a sequence specific transcription
  factor on its own.
• E in humans 5S rRNA genes are arranged in a
  single cluster of 2000 copies.

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• 5. Which one of the following statements is
  true?
• A RNA Pol II only transcribes protein-coding
  genes.
• B the TATA box has a role in transcription
  efficiency but not in positioning the start of
  transcriphon.
• C TBP binds to the TAT A box.
• D Enhancers typically lie 100-200 bp upstream
  from the start of transcription.
• 6. Which one of the following statements about
  general transcription factors is false?
• A TFIID binds to the T ATA box.
• B TFIID is a multi protein complex consisting
  of TBP and TAFIIs.
• C TBP is a common factor in transcription by
  RNA Pol I, RNA Pol II and RNA Pol III.
• D TFIIB stabilizes the TFIID-DNA complex.
• E TFIIE, TFIIH and TFIIJ associate with the
  transcription complex after RNA polymerase
  binding.
• F TFIIH phosphorylates the CTD.

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