Transcript M2 RNA Pol Ⅰ genes

Section M – Transcription in
eukaryotes
Contents
M1 The three RNA polymerases:
characterization and function
Eukaryotic RNA polymerase, RNA polymerase subunits, Eukaryotic
RNA polymerase activities, The CTD of RNA PolⅡ
M2 RNA Pol Ⅰ genes: the ribosomal repeat
Ribosomal RNA genes, Role of the nucleolus, RNA PolⅠ promoters,
Upstream binding factor, Selectivity factor 1, TBP and TAF1s, Other
rRNA genes
M3 RNA Pol Ⅲ genes: 5s and tRNA transcription
RNA polymerase Ⅲ, tRNA genes, 5s rRNA genes, Alternative RNA
PolⅢ promoters, RNA PolⅢ termination
M4 RNA Pol Ⅱ genes: promoters and enhancers
RNA polymerase Ⅱ, Promoters, Upstream regulatory elements,
Enhancers
M5 General transcriptiion factors and RNA Pol
Ⅱ initiation
RNA PolⅡ basal transcription factors, TFⅡD, TBP, TFⅡA, TFⅡB and
RNA polymerase binding, Factors binding after RNA polymerase,
CTD phosphorylation by TFⅡH, The initiator transcriptiom complex
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(毒素) α-amanitin (α-鹅膏
蕈碱).
• RNA polymerase I is located in the nucleoli. It’s
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.It’s
responsible foe the synthesis of the precursors of 5s
rRNA, tRNAs and other small nuclear and cytosolic(细胞
溶质的) RNAs.
Three eukaryotic polymerases
Type
Location Substrate
a-amanitin
RNA Pol I
Nucleoli
Most rRNAs gene
Insensitive
RNA Pol II
Nucleoplasm
All protein-coding Very
genes and some
sensitive
snRNA genes
RNA Pol III
Nucleoplasm
tRNAs, 5S rRNA,
U6 snRNA and
other small RNAs
Moderately
sensitive
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.
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.
M1 The three RNA polymerases: characterization
and function —
•
•
•
•
The CTD of RNA PolⅡ
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-ProThr-Ser-Pro-Ser
This repeated sequence is known as
carboxyl terminal domain (CTD)
The CTD sequence may be phosphorylated
at the serines and some tyrosines
• 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.
M2 RNA Pol Ⅰ 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.
3. Continuous transcription of multiple copies of
rRNA genes by RNA Pol I is essential to
produce sufficient rRNAs which are packaged
into ribosomes.
4. The arrays of rRNA genes (rRNA cluster) loop
together to form the nucleolus and are known
as nucleolar (核仁的)organizer regions.
5. During active rRNA synthesis, the pre-rRNA
transcripts are packaged along the rRNA genes,
visualizing in the electronic microscope as
“Christmas tree structures”.
M2 RNA Pol Ⅰ 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.
We should clearly understand what a
promoter is before further study ?
Let’s review the concept.
A promoter is the site on the DNA
to which an RNA polymerase
molecule binds prior to initiating
transcription.
Antisense or template DNA
strand : the strand from which
it copies.
Sense or coding strand:
the other strand, to which it is
identical RNA.
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.
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).
Prokaryotic promoter sequences
-10 position
( called Pribnow
box,TATAAT)
-35 position
(TTGACA)
Be absolutely
essential to start
transcription in
prokaryotes.
Allows a very high
transcription rate.
Then ,we can know that the
bacterial promoter almost always
contains some version of the
following elements:
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)
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.
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.
M2 RNA Pol Ⅰ genes: the ribosomal repeat —
RNA PolⅠ 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 G:C, with ~85% identity.
M2 RNA Pol Ⅰ 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
M2 RNA Pol Ⅰ 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.
M2 RNA Pol Ⅰ 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.
The initiation complex assembles in three stages
M2 RNA Pol Ⅰ 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.
M3 RNA Pol Ⅲ genes: 5s and tRNA transcription —
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).
M3 RNA Pol Ⅲ 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).
• 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.
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”.
TFIIIC: A and B boxes
binding and a assembly
factor to position TFIIIB
TFIIIB: DNA binding and
RNA Pol III recruiting
M3 RNA Pol Ⅲ 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
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.
M3 RNA Pol Ⅲ genes: 5s and tRNA transcription —
Alternative RNA PolⅢ 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.
U6 snRNA
1. The coding region contains a characteristic A
box that is not required for transcription.
2. The upstream sequence contains sequences
typical of RNA Pol II promoters, including a
TATA box at bases –30 to –23.
3. 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
M3 RNA Pol Ⅲ genes: 5s and tRNA transcription —
RNA PolⅢ 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’
M4 RNA Pol Ⅱ genes: promoters and enhancers —
RNA polymerase Ⅱ
•
•
•
located in nucleoplasm
catalyzing the synthesis of the mRNA
precursors for all protein-coding
genes.
RNA Pol Ⅱ-transcribed pre-mRNAs
are processed through cap addition,
poly(A) tail addition and splicing.
M4 RNA Pol Ⅱ 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.
•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.
M4 RNA Pol Ⅱ 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.
M4 RNA Pol Ⅱ genes: promoters and enhancers —
Enhancers
Enhancers :
• Sequence elements which can activate
transcription from thousands of base
pairs upstream or downstream.
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
M5 General transcriptiion factors and RNA Pol Ⅱ
initiation —
RNA PolⅡ 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.
M5 General transcriptiion factors and RNA Pol Ⅱ
initiation —
TFⅡD
• Multiprotein Complex, including TBP,
other proteins are known as TAFIIs.
• TBP is the only protein binds to TATA
box
M5 General transcriptiion factors and RNA Pol Ⅱ
initiation —
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.
TBP
Outer surface (with ?)
TBP
DNA
Inner surface (with ?)
M5 General transcriptiion factors and RNA Pol Ⅱ
initiation —
TFIIA
• binds to TFIID
• stabilizes TFIID-DNA complex
• contains at least 3 subunits
TFⅡA
M5 General transcriptiion factors and RNA Pol Ⅱ
initiation —
TFⅡB and RNA polymerase
binding
• TFIIB & RNA Pol
binding
• binds to TFIID
• Binds to RNA Pol
with TFIIF
M5 General transcriptiion factors and RNA Pol Ⅱ
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.
4-1 TFIIE binding
•Necessary for
transcription
4-2 TFIIJ, TFIIH
binding
•Necessary for
transcription
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.
M5 General transcriptiion factors and RNA Pol Ⅱ
initiation —
CTD phosphorylation by
TFⅡH
• TFIIH phosphorylates(使磷酸化) the
carboxy-terminal domain (CTD，羟基末端
结构域) of RNA Pol II.
• This results in formation of a processive
polymerase complex.
M5 General transcriptiion factors and RNA Pol Ⅱ
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.
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 toα-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.
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.
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.
THANK YOU !