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Section M Transcription in Eukaryotes

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The three RNA Polymerases: characterization and function. M2. ... Xenopus borealis terminator: 5'-GCAAAAGC-3' M4. RNA Pol II genes: promoters and enhancers ... – PowerPoint PPT presentation

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Title: Section M Transcription in Eukaryotes


1
Section M Transcription in Eukaryotes
  • Molecular Biology Course

2
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
3
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

4
Section M Transcription in Eukaryotes
M1-1 Eukaryotic RNA polymerases
5
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.

6
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.
7
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
9
  • 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.

10
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11
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.

12
Section M Transcription in Eukaryotes
M1-4 The CTD of RNA pol II
13
  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

14
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.
15
Section M Transcription in Eukaryotes
M2. RNA Pol I genes the ribosomal repeats
16
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
17
Section M Transcription in Eukaryotes
M2-12 Structure of the rRNA genes 1.
Ribosomal RNA genes 2. Role of the necleolus
18
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.

19
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.

21
Christmas Tree Structures
22
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
23
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.

24
RNA Pol I promoters in human cells
Your text shows RNA Pol I promoter from another
organism
25
Two ancillary factors (UBF SL1) of RNA Pol I
their roles in transcription initiation
Section M Transcription in Eukaryotes
26
  • 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.

27
  • 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.

28
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.

29
  • The initiation complex assembles in three stages

30
The initiation complex proposed in your text
book
It is not known which representation one is more
accurate.
31
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.
32
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

33
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

34
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).
35
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36
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).

37
  • 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.

38
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.
39
TFIIIC A and B boxes binding and a assembly
factor to position TFIIIB
TFIIIB DNA binding and RNA Pol III recruiting
40
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

41
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.
42
TFIIIA
TFIIIC
TFIIIB
Pol III
43
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.

44
  • 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

45
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
46
Section M Transcription in Eukaryotes
M4. RNA Pol II genes promoters and enhancers
  • RNA Pol II
  • Cis-acting elements
  • Promoters
  • Upstream regulatory elements
  • Enhancers

47
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.

48
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.

49
  • 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.

50
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.
51
Section M Transcription in Eukaryotes
M4-3. Upstream regulatory elements
52
  • 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.

53
Section M Transcription in Eukaryotes
M4-4. Enhancers
Sequence elements which can activate
transcription from thousands of base pairs
upstream or downstream.
54
  • 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

55
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

56
1. TFIID Multiprotein Complex, including TBP,
other proteins are known as TAFIIs. TBP is the
only protein binds to TATA box
57
TBP 1. a general transcription factor bound to
DNA at the TATA box. 2. a general transcription
required by all 3 RNA pol.
58
TBP 3. Has a saddle structure with an overall
dyad symmetry.
Outer surface (with ?)
TBP
DNA
Inner surface (with ?)
59
TBP
45o Kink
TBP causes DNA bending
60
  • 2. TFIIA
  • binds to TFIID
  • stabilizes TFIID-DNA complex
  • contains at least 3 subunits

61
  • 3. TFIIB RNA Pol binding
  • binds to TFIID
  • Binds to RNA Pol with TFIIF

62
  • 4-1 TFIIE binding
  • Necessary for transcription

63
  • 4-2 TFIIJ, TFIIH binding
  • Necessary for transcription

64
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.
65
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.
66
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