Section M Transcription in Eukaryotes

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Section M Transcription in Eukaryotes

• Molecular Biology Course

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Section M Transcription in Eukaryotes
Topics M1. The three RNA Polymerases
characterization and function M2. RNA Pol I
genes the ribosomal 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
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Section M Transcription in Eukaryotes
M1. The three RNA Polymerases characterization
and function

1. Eukaryotic RNA polymerases
2. RNA polymerase subunits
3. Eukaryotic RNA polymerase activities
4. The CTD of RNA Pol II

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Section M Transcription in Eukaryotes
M1-1 Eukaryotic RNA polymerases
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Section M Transcription in Eukaryotes
Main Features of eukaryotic transcription

1. The mechanism of eukaryotic transcription is
   similar to that in prokaryotes.
2. A lot more proteins are associated with the
   eukaryotic transcription machinery, which results
   in the much more complicated transcription.

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Section M Transcription in Eukaryotes
3. Three eukaryotic polymerases transcribe
different sets of genes. The activities of these
polymerases are distinguished by their
sensitivities to the fungal toxin a-amanitin
(????). 4. In addition, eukaryotic cells contain
additional RNA Pols in mitochondria and
chloraplasts.
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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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Section M Transcription in Eukaryotes
M1-2 RNA polymerase subunits
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• 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.
• There is one other subunit in all three RNA Pol
  homologous to a subunit of E. coli RNA Pol.
• Five additional subunits are common to all three
  polymerases.
• Each RNA Pol contain additional four or seven
  specific subunit.

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Section M Transcription in Eukaryotes
M1-3 RNA polymerase activities

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

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Section M Transcription in Eukaryotes
M1-4 The CTD of RNA pol II
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1. 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.
2. The heptapeptide sequenc is Tyr-Ser-Pro-Thr-Ser-P
   ro-Ser
3. This repeated sequence is known as carboxyl
   terminal domain (CTD)
4. 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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Section M Transcription in Eukaryotes
M2. RNA Pol I genes the ribosomal repeats
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Section M Transcription in Eukaryotes
1-2 Structure of the rRNA genes 1. Ribosomal
RNA genes 2. Role of the necleolus 3-6RNA Pol I
promoters binding factors 3. RNA Pol I
promoters 4. Upstream binding factor (UBF) 5.
Selectivity factor 1 6. TBP and TAFIs 7 Other
rRNA genes
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Section M Transcription in Eukaryotes
M2-12 Structure of the rRNA genes 1.
Ribosomal RNA genes 2. Role of the necleolus
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Ribosomal RNA Genes nucleolus

1. 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.
2. Pre-rRNA transcription units are arranged in
   clusters in the genome as long tandem arrays
   separated by nontranscribed spacer squences.

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A single transcription unit
Tandem array
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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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Christmas Tree Structures
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Section M Transcription in Eukaryotes
M2-36 RNA Pol I promoters binding factors 3.
RNA Pol I promoters 4. Upstream binding factor
(UBF) 5. Selectivity factor 1 6. TBP and TAFIs
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RNA Pol I 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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RNA Pol I promoters in human cells
Your text shows RNA Pol I promoter from another
organism
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Two ancillary factors (UBF SL1) of RNA Pol I
their roles in transcription initiation
Section M Transcription in Eukaryotes
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• Upstream binding factor (UBF)
• 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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• Selectivity factor 1 (SL1)
• 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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Subunits of SL1

• 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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The initiation complex proposed in your text
book
It is not known which representation one is more
accurate.
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Other rRNA genes (simple)
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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Section M Transcription in Eukaryotes
M3. RNA Pol III genes 5S and tRNA transcription

1. RNA polymerase III
2. tRNA genes
3. 5S rRNA genes
4. Alternative RNA Pol III promoters
5. RNA Pol III termination

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Section M Transcription in Eukaryotes
M3-1. RNA Pol III

1. Contains at least 16 or more subunits
2. Is located in nucloplasm
3. Synthesizes the precursors of 5S rRNA, the tRNAs
   and other small nuclear and cytosolic RNAs

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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).
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Section M Transcription in Eukaryotes
M3-2. tRNA genes

1. The initial transcripts of tRNA genes need to be
   processed to produce the mature tRNA.
2. 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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Section M Transcription in Eukaryotes
M3-3. 5S rRNA genes

1. Tandemly arranged in a gene cluster. (In human,
   there is a single cluster of around 2000 genes.)
2. 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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TFIIIA
TFIIIC
TFIIIB
Pol III
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Section M Transcription in Eukaryotes
M3-4. Alternative RNA Pol III 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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Section M Transcription in Eukaryotes
M3-5. RNA Pol III 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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Section M Transcription in Eukaryotes
M4. RNA Pol II genes promoters and enhancers

• RNA Pol II
• Cis-acting elements
• Promoters
• Upstream regulatory elements
• Enhancers

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Section M Transcription in Eukaryotes
M4-1. RNA Pol II

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

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Section M Transcription in Eukaryotes
M4-2. 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.

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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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Section M Transcription in Eukaryotes
M4-3. Upstream regulatory elements
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• 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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Section M Transcription in Eukaryotes
M4-4. 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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Section M Transcription in Eukaryotes
M5. General transcription factors and RNA Pol?
initiation

• RNA Pol II basal transcription factors
• TFIID (TBP)
• 3. TFIIA
• 4. TFIIB and RNA Pol binding
• 5. Factors binding after RNA Pol.
• 6. CTD phosphorylation by TFIIH
• 7. The initiator complex

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1. TFIID Multiprotein Complex, including TBP,
other proteins are known as TAFIIs. TBP is the
only protein binds to TATA box
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TBP 1. a general transcription factor bound to
DNA at the TATA box. 2. a general transcription
required by all 3 RNA pol.
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TBP 3. Has a saddle structure with an overall
dyad symmetry.
Outer surface (with ?)
TBP
DNA
Inner surface (with ?)
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TBP
45o Kink
TBP causes DNA bending
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• 2. TFIIA
• binds to TFIID
• stabilizes TFIID-DNA complex
• contains at least 3 subunits

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

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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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The initiator transcription 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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Thanks