Transcript Transcription

Chapter 5
Transcription
A. Transcription in
prokaryotes
5.1 Basic principles of transcription
An overview, the process of RNA synthesis
( initiation, elongation, termination)
5.2 Escherichia coli RNA polymerase
Properties, a subunit, b subunit, b’ subunit,
sigma (s) factor
5.3 The E. coli s70 promoter
Promoter, s70 size, -10 sequence, -35 sequence,
transcription start site, promoter efficiency
5.4 transcription process.
Promoter binding, unwinding, RNA chain
initiation, elongation, termination (r factor)
5.1: Basic principles of
transcription
1. Transcription: an overview
(comparison with replication)
2. The process of RNA synthesis:
initiation, elongation, termination
5.1-1: Transcription: an overview
Key terms defined in this section
(Gene VII)
+1
upstream
Gene X
downstream
Primary transcript
m7Gppp
AAAAAn
mRNA
Coding strand of DNA has the same sequence
as mRNA.
Downstream identifies sequences proceeding
further in the direction of expression; for
example, the coding region is downstream of
the initiation codon.
Upstream identifies sequences proceeding in
the opposite direction from expression; for
example, the bacterial promoter is upstream
from the transcription unit, the initiation
codon is upstream of the coding region.
Transcription unit is the distance between
sites of initiation and termination by RNA
polymerase; may include more than one gene.
Promoter is a region of DNA involved in
binding of RNA polymerase to initiate
transcription
RNA Terminator is a sequence of DNA,
represented at the end of the transcript, that
causes RNA polymerase to terminate
transcription.
RNA polymerases are enzymes that
synthesize RNA using a DNA template
(formally described as DNA-dependent RNA
polymerases).
Primary transcript is the original unmodified
RNA product corresponding to a transcription
unit.
Replication: synthesis of two DNA
molecules using both parental
DNA strands as templates.
Duplication of a DNA molecule.
1 DNA molecule  2 DNA molecules
Transcription: synthesis of one
RNA molecule using one of the two
DNA strands as a template.
1 DNA molecule  1 RNA molecule
Replication-synthesis of the
leading strand
the same direction as the replication fork moves
Replication- Synthesis of the
Okazaki fragments
Opposite to the replication fork movement
Coupling the synthesis of leading and
lagging strands with a dimeric DNA pol
III (E. coli)
Transcription
1. RNA synthesis occurs in the 5’3’
direction and its sequence corresponds
to the sense strand (coding strand).
2. The template of RNA synthesis is the
antisense strand (template strand).
3. Phosphodiester bonds: same as in DNA
4. Necessary components: RNA
polymerase, transcription factors, rNTPs,
promoter & terminator/template
DNA 非模板链（编码链）,
（+）正链 或正义链）
DNA 双螺旋
5’-CGCTATAGCGTTTGCAGGCGTTCACGGC-3’
3’-GCGATATCGCAAACGTCCGCAAGTGCCG-5’
转录
模板链,负链（-）
或反义链
mRNA （RNA transcript）
5’-CGCUAUAGCGUUUGCAGGCGUUCACGGC-3’
5.1-2: The process of RNA
synthesis
1.initiation
2.elongation
3.termination
+1
Promoter
DNA
Terminator
Sense
strand
Transcribed region
Transcription
Antisense strand
RNA
Fig. 2. Structure of a typical transcription unit
Before initiation: RNA pol recognizes
promoter (RNA聚合酶识别启动子)
1. RNA聚合酶结合到启动子上游附近的
双链DNA模板；
2. 沿双链DNA滑动，找到启动子
(promoter)序列；
3. 负责识别启动子的是RNA聚合酶的σ
亚基；
4. 真核生物的转录在启动子(promoter)
序列处首先结合转录起始因子；
8-18
Initiation (template recognition)
1. Binding of an RNA polymerase to the
dsDNA
2. Slide to find the promoter
3. Unwind the DNA helix
4. Synthesis of the RNA strand at the
start site (initiation site), this position
called position +1
Link
Elongation
• Covalently adds ribonucleotides to the
3’-end of the growing RNA chain.
• The RNA polymerase extend the
growing RNA chain in the direction of
5’ 3’
• The enzyme itself moves in 3’ to 5’ along
the antisense DNA strand.
Link
Termination
• Ending of RNA synthesis: the
dissociation of the RNA polymerase and
RNA chain from the template DNA at
the terminator site.
• Terminator: often contains selfcomplementary regions which can form
a stem-loop or hairpin structure in the
RNA products
Terminator structure
5.2 Escherichia coli RNA
polymerase
1. E. coli RNA polymerase
2. a subunit
3. b subunit
4. b’ subunit
5.sigma (s) factor
5.2-1 E. coli RNA polymerase
Synthesis of single-stranded RNA
from DNA template.
RNA polymerase
(NMP)n + NTP  (NMP)n+1 + PPi
1. Requires no primer for polymerization
2. Requires DNA for activity and is most active
with a double-stranded DNA as template.
3. 5’  3’ synthesis
4. Require Mg2+ for RNA synthesis activity
5. lacks 3’  5’ exonuclease activity, and the
error rate of nucleotides incorporation is 10-4
to 10-5. Is this accuracy good enough for gene
expression??
6. usually are multisubunit enzyme.
E. coli polymerase
1. E. coli has a single DNA-directed RNA
polymerase that synthesizes all types of RNA.
2. One of the largest enzyme in the cells
3. Consists of at least 5 subunits in the
holoenzyme, 2 alpha (a), and 1 of beta (b), beta
prime (b’), omega (w) and sigma (s) subunits
4. Shaped as a cylindrical channel that can bind
directly to 16 bp of DNA. The whole
polymerase binds over 60 bp.
5. RNA synthesis rate: 40 nt per second at 37oC
E. coli RNA polymerase
155 KD
36.5 KD
11 KD
36.5 KD
70 KD
151 KD
Both initiation & elongation
Initiation only
E.coli RNA 聚合酶的亚基性质和功能
亚基
基因 相对分子量 亚基数
a
rpoA
4.0104
2
core
b
rpoB
1.51105
1
core
b’
w
s
rpoC
?
rpoD
1.55105
11104
7.0104
1
1
1
功能
core assemble,
promoter recognition
b and b’combined
together to form
catalyzed center
core
core
unknown
s factor different factors
recognize different
8-28
promoters
可解离的sigma亚基赋予RNA聚合酶
对原核启动子的特异性
a
a
b
b’
+
w
s
a s b
a
b’
w
核心酶
Core enzyme
全酶
Holoenzyme
非特异性结合启动
子，并且结合紧密
特异性结合启动子，
结合程度较弱
8-29
RNA聚合酶起始转录
全酶
“扫描”
a s b
a
b’
封闭复合物
rNTPs
启动子
-35
-10
a s b
a
b’
PPi
开放复合物; 起始
5’pppA
核心酶
a
a
mRNA
b
b’
s
8-30
RNA聚合酶的主要功能
①识别和结合DNA链上的启动子；
②能沿DNA双链作单向运动；
③解开DNA双螺旋，转录后又恢复双螺旋；
④能同时结合单链DNA和转录产物RNA；
⑤按DNA反义链为模板选择正确的底物NTP，
以5’ → 3’方向催化磷酸二酯键的形成，合
成RNA链；
⑥识别转录的终止信号；
⑦能够与转录因子相互作用，调节转录；
⑧能在转录受到阻遏时进行自我调整，借助
辅助因子，恢复和维持RNA的合成。 8-31
The polymerases of bacteriophage T3
and T7 are smaller single polypeptide chains,
they synthesize RNA rapidly (200 nt/sec) and
recognize their own promoters which are
different from E. coli promoters.
RNA polymerase differs from
organism to organism
5.2-2: a subunit
E. coli polymerase: a subunit
•
•
•
•
Two identical subunits in the core enzyme
Encoded by the rpoA gene
Required for assembly of the core enzyme
Plays a role in promoter recognition.
Experiment: When phage T4 infects E. coli, the
α subunit is modified by ADP-ribosylation of an
arginine. The modification is associated with a
reduced affinity for the promoters formerly
recognized by the holoenzyme.
• plays a role in the interaction of RNA
polymerase with some regulatory factors
大肠杆菌RNA聚合酶: a 亚基
1. 在核心酶中两个a亚基是相同的；
2. a亚基是由rpoA 基因编码；
3. 对于RNA聚合酶核心蛋白的组装是
必需的；
4. 在启动子识别上可能起着重要的作
用；
5.2-3&4: b and b’ subunit
1. b is encoded by rpoB gene, and b’ is encoded
by rpoC gene .
2. Make up the catalytic center of the RNA
polymerase
3. Their sequences are related to those of the
largest subunits of eukaryotic RNA polymerases,
suggesting that there are common features to
the actions of all RNA polymerases.
4. The b subunit can be crosslinked to the
template DNA, the product RNA, and the
substrate ribonucleotides; mutations in rpoB
affect all stages of transcription. Mutations in
rpoC show that b’ also is involved at all stages.
b subunit may contain two domains
responsible for transcription initiation and
elongation
•Rifampicin (利福平)：has been shown to bind
to the β subunit, and inhibit transcription
initiation by prokaryotic RNA pol. Mutation in
rpoB gene can result in rifampicin resistance.
•Streptolydigins(利迪链菌素)：resistant
mutations are mapped to rpoB gene as well.
Inhibits transcription elongation but not
initiation.
b’ subunit
•
Binds two Zn 2+ ions and may participate in
the catalytic function of the polymerase
• Hyparin (肝素)：binds to the b’ subunit and
inhibits transcription in vitro.
• Hyparin competes with DNA for binding to the
polymerase.
2. b’ subunit may be responsible for binding to
the template DNA .
大肠杆菌RNA聚合酶: b 亚基
1. β亚基由rpoB 基因编码；
2. RNA聚合酶的催化中心；
Rifampicin (利福平)：与β亚基结合可以抑
制转录的起始，rpoB 基因突变导致对利福平
的抗性；
Streptolydigins(利迪链菌素)：抗性突变也
定位于 rpoB 基因，它抑制转录延伸，但不抑
制起始；
3. β亚基可能含有两个结构域负责转录的起始和
延伸；
大肠杆菌RNA聚合酶: b’亚基
1. b 亚基由rpoC 基因编码；
2. 与两个Zn2+ 离子结合参与RNA聚合酶的
催化功能；
• Heparin (肝素)：在体外与 β’ 亚基结合
并抑制转录；
• Heparin 与DNA竞争结合RNA聚合酶；
3. β’ 亚基可能是负责与DNA模板的结合；
5.2-5: Sigma (s) factor
1. Many prokaryotes contain multiple s factors
to recognize different promoters. The most
common s factor in E. coli is s70.
2. Binding of the s factor converts the core RNA
pol into the holoenzyme.
3. s factor is critical in promoter recognition, by
decreasing the affinity of the core enzyme for
non-specific DNA sites (104) and increasing
the affinity for the corresponding promoter
4. s factor is released from the RNA pol after
initiation (RNA chain is 8-9 nt)
5. Less amount of s factor is required in cells
than that of the other subunits of the RNA pol.
大肠杆菌RNA聚合酶: s亚基
1. 在启动子的识别上σ 因子至关重要，对
于非特异性DNA位点核心酶的亲和力
会降低（104），而对于相应的启动子
亲和力会增加；
2. 当起始后(RNA chain is 8-9 nt) ，σ 因子
会从RNA聚合酶中释放出来；
3. 在细胞中σ 因子的需要量比RNA聚合酶
其他亚基的需要量少；
5.3: The E. coli s70 promoter
1. Promoter
2. s70 size
3. -10 sequence
4. -35 sequence
5. transcription start
site
6. promoter efficiency
5.3-1: Promoter
1. The specific short conserved DNA sequences:
2. upstream from the transcribed sequence, and
thus assigned a negative number (location)
3. required for specific binding of RNA Pol. and
transcription initiation (function)
4. Were first characterized through mutations
that enhance or diminish the rate of
transcription of gene
+1
Promoter
DNA
Terminator
Transcribed region Sense strand
Transcription
Antisense strand
RNA
Different promoters result in differing
efficiencies of transcription initiation,
which in turn regulate transcription.
5.3-2,3&4: s70 promoter
G
TTGACA-----16-18 bp------- TATAAT ---5-8 bp--- C T
A
-35 sequence
-10 sequence
+1
• Consists of a sequence of between 40 and 60 bp
• -55 to +20: bound by the polymerase
• -20 to +20: tightly associated with the
polymerase and protected from nuclease
digestion by DNaseΙ(see the supplemental)
• Up to position –40: critical for promoter
function (mutagenesis analysis)
• -10 and –35 sequence: 6 bp each, particularly
important for promoter function in E. coli
-10 sequence (Pribonow box)
1. The most conserved sequence in s70
promoters at which DNA unwinding is
initiated by RNA Pol.
2. A 6 bp sequence which is centered at around
the –10 position, and is found in the
promoters of many different E. coli gene.
3. The consensus sequence is TATAAT. The
first two bases (TA) and the final T are most
highly conserved.
4. This hexamer is separated by between 5 and
8 bp from position +1, and the distance is
critical.
-35 sequence: enhances recognition
and interaction with the polymerase s
factor
• A conserved hexamer sequence around
position –35
• A consensus sequence of TTGACA
• The first three positions (TTG) are the
most conserved among E. coli promoters.
• Separated by 16-18 bp from the –10 box
in 90% of all promoters
It can increase to
recognize RNA Polyσ
核心启动子区包括
Sextama Box ;
-35 区 RNA聚合酶松散结合位点
RNA聚合酶的识别位点
(R to
site)
It is crucial
loose
DNA helix
TTGAC (Sextama Box)
Pribnow Box ;
-10 区 RNA聚合酶紧密结合位点 (B site)
TATAAT (pribnow Box)
Initiation site ; +1 RNA transcriptional startpoint (I site)
A/G
RNA PolyRNA Poly
-35 (R)
-10 (B)
RNA Poly
+1 (I)
RNA
8-53
The best interval between -10~-35 region
In prokaryote, the interval between -10~-35 region
is about 16~19bp. Promoter activity will be
decreased while the interval is <15bp or >20bp. 8-54
RNA聚合酶在DNA链上的构型变化
1.全酶与DNA接触时占据
长度为75~80bp，从-55 ~
+20；
2. 进入延伸阶段， 伴随σ
因子的释放，构象发生变
化，覆盖的长度为60 bp;
3. 当新生RNA链聚合15~20bp时，构象进一步发生
转变，形成延伸反应复合物，此时覆盖的长度为
30~40bp的DNA;
Supplemental material
RNA Polymerase Leaves Its
FootPrint on a Promoter
• Footprinting is a
technique derived
from principles used
in DNA sequencing.
It is used to identify
the specific DNA
sequences that are
bound by a
particular protein.
Footprinting
Footprinting
5.3-5: Transcription start site
• Is a purine in 90% of all gene
• G is more common at position +1 than A
• There are usually a C and T on either
side of the start nucleotide (i.e. CGT or
CAT)
The sequences of five E. coli promoters
K3-6: promoter efficiency
There is considerable variation in sequence
between different promoters, and the
transcription efficiency can vary by up to
1000-fold .
1. The –35 sequence constitutes a recognition
region which enhances recognition and
interaction with the polymerase s factor.
2. The -10 sequence is important for DNA
unwinding.
3. The sequence around the start site influence
initiation efficiency.
4. The sequence of the first 30 bases to be
transcribed controls the rate at which the
RNA polymerase clears the promoter, hence
influences the rate of the transcription and the
overall promoter strength.
Weak promoters and activating factor
Some promoter sequence are not sufficiently
similar to the consensus sequence to be strongly
transcribed under normal condition, thus
activating factor is required for efficient
initiation.
Example: Lac promoter P lac requires activating
protein, cAMP receptor protein (CRP ), to bind
to a site on the DNA close to the promoter
sequence in order to enhance polymerase binding
and transcription initiation.
5.4 Transcription process
1. Promoter binding
2.DNA unwinding
3.RNA chain initiation
4.RNA chain elongation
5.RNA chain termination (r
factor)
1. Promoter binding
The searching process is
extremely rapidly
Closed complex:
the initial complex
of the polymerase
with the base-paired
promoter DNA)
and –10 region
Link
The role of s factor in promoter binding
• The RNA polymerase core enyzme, a2bb’w, has
a general non-specific affinity for DNA, which is
referred to as loose binding that is fairly stable.
• The addition of s factor to the core enzyme
markedly reduces the holoenzyme affinity for
non-specific binding by 20 000-fold, and
enhances the holoenzyme binding to correct
promoter sites 100 times.
• Overall, s factor binding dramatically increases
the specificity of the holoenzyme for correct
promoter-binding site.
2. DNA unwinding
+1
The initial unwinding of the DNA results in
formation of an open complex with the
polymerase, and this process is referred to as
tight binding
Negative supercoiling & unwinding
• It is necessary to unwind the DNA so that the
antisense strand to become accessible for base
pairing and RNA synthesis.
• Negative supercoiling enhances the
transcription of many genes, since it facilitates
unwinding. Some promoters are not.
• Exceptional example: promters for the enzyme
subunits of DNA gyrase are inhibited by
negative supercoiling, serving as an elegant
feedback loop for DNA gyrase expression.
3. RNA chain initiation
Starts with a GTP or ATP
+1
The polymerase initially incorporates the first
two nucleotides and forms a phosphodiester bond.
Abortive initiation
The first 9 nt are incorporated without
polymerase movement along the DNA.
Afterward, there is a significant
probability that the chain will be aborted.
• The RNA pol. goes through multiple abortive
initiations before a successful initiation, which
limits the overall rate of transcription
• The minimum time for promoter clearance is
1-2 seconds (a long event, the synthesis is 40 nt/
sec)
4. RNA chain elongation
• Promoter clearance: when initiation
succeeds, the enzyme releases s factor
and forms a ternary complex of
polymerase-DNA-nascent RNA, causing
the polymerase to progress along the
DNA to allow the re-initiation of
transcription.
Transcription bubble:
1. containing ~ 17 bp of unwound DNA
region and the 3’-end of the RNA that
forms a hybrid helix about 12 bp.
2. moves along the DNA with RNA
polymerase which unwinds DNA at the
front and rewinds it at the rear.
3. The E. coli polymerase moves at an
average rate of ~ 40 nt per sec,
depending on the local DNA sequence.
Transcription bubble
5. RNA chain termination
1.Termination occurs at terminator DNA
sequences.
2.The most common stop signal is an RNA
hairpin (self-complement structure)
commonly GC-rich to favor the structure
stability
3. Rho-dependent and Rho-independent
Termination.
Terminator
A specific DNA sequence where the
transcription complex dissociate
Rho protein (r) independent terminator contains:
(1) self-complementary region that is G-C rich
and can form a stem-loop or hairpin secondary
structure. GC-rich favouring the base pairing
stability and causing the polymerase to pause.
(2) a
run of adenylates (As) in the template strand
that are transcribed into uridylates (Us) at the
end of the RNA, resulting in weak RNAantisense DNA strand binding.
A model for rindependent termination
of transcription in E. coli.
The A-U base-pairing is
less stable that favors the
dissociation
Rho protein (r) dependent terminator
1.
2.
3.
4.
Contains only the self-complementary region
Requires r protein for termination
r protein binds to specific sites in the singlestranded RNA
r protein hydrolyzes ATP and moves along the
nascent RNA towards the transcription
complex then enables the polymerase to
terminate transcription
RNA polymerase/transcription
and DNA polymerase/replication
RNA pol
DNA pol
dsDNA
Require primer
dsDNA is better
than ssDNA
No
Initiation
promoter
origin
elongation
40 nt/ sec
900 bp/sec
terminator
Synthesized RNA Template DNA
Template
Yes
In any chromosome, different genes may use different
strands as template (Fig. 25-2).
B. Transcription in
Eukaryotes
原核与真核基因转录的差异
1）只有一种RNA聚合酶参与原核基因的转录，
真核生物有3种以上的RNA聚合酶负责不同
类型的基因转录。
2）转录产物差别很大，以多顺反子形式存在，
真核转录产物是以单顺反子形式存在成熟
的mRNA只占初始转录产物的一小部分。
3）真核的转录产物需要经过剪接，加工成熟，
而原核转录产物几乎不需要加工。
4）原核转录产物为多顺反子，大多数真核
生物的mRNA是单顺反子；
5）在原核生物细胞中，转录产物可以直接
作为蛋白质合成的模板，转录mRNA与
蛋白质的翻译相互偶联。真核生物细胞
的转录是在细胞核进行的成熟后通过核
孔进入细胞质在此翻译出蛋白质。
5.5 The three RNA Polymerases:
characterization and function
5.6 RNA Pol I genes: the ribosomal
repeat
5.7 RNA Pol III genes: 5S and
tRNA transcription
5.8 RNA Pol II genes: promoters
and enhancers
5.9 General transcription factors
and RNA Pol II initiation
5.5 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
5.5-1:
Eukaryotic RNA polymerases
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.
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 (鹅膏菌素,
或鹅膏蕈碱).
4. In addition, eukaryotic cells
contain additional RNA Pols in
mitochondria and chloraplasts.
Three eukaryotic polymerases
Type
Location Substrate
a-amanitin
RNA Pol I
Nucleoli
Insensitive
Most rRNAs gene
RNA Pol II Nucleoplasm
All protein-coding Very
genes and some
sensitive
snRNA genes
RNA Pol
III
tRNAs, 5S rRNA,
U6 snRNA and
other small RNAs
Nucleoplasm
Moderately
sensitive
真核RNA聚合酶活性
Eukaryotic RNA polymerase activity
1）Similarities to that in prokaryotic
cells
1. Don’t require a primer
2. Synthesize RNA in a 5’ to 3’
direction.
3. RNA complementary to the
antisense template strand.
2） Difference to bacterial polymerases
1. There are 3 RNA polymerases
2. Require more accessory factors for binding
promoter DNA & initiating transcription.
3. The C-terminus of RNA Pol II largest subunit
contains a stretch of heptapeptide repeats,
named as carboxyl terminal domain (CTD)
• Amino acid sequence: Tyr-Ser-Pro-Thr-SerPro-Ser. Repeated 26 x (yeast) & 52x in mouse
• Involved in polymerase phosphorylation
during elongation.
4. The CTD is unphosphorylated at
transcription initiation, and
phosphorylation occurs during
transcription elongation as the RNA
Pol II leaves the promoter (In vitro
results).
5. 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.
5.5-2:
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.
– 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.
5.5-3:
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.
5.5-4:
The CTD of RNA pol II
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-SerPro-Thr-Ser-Pro-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
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.
5.6 RNA Pol I genes:
the ribosomal repeats
1-2: Structure of the rRNA genes
1. Ribosomal RNA genes
2. Role of the necleolus
3-6:RNA 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
5.6-1&2: Structure of the
rRNA genes
1. Ribosomal RNA genes
2. Role of the necleolus
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.
A single transcription unit
Tandem array
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
organizer regions.
5. During active rRNA synthesis, the prerRNA transcripts are packaged along
the rRNA genes, visualizing in the
electronic microscope as “Christmas
tree structures”.
Christmas
Tree Structures
5.6-3~6: RNA Pol I promoters &
binding factors
3. RNA Pol I promoters
4. Upstream binding factor
(UBF)
5. Selectivity factor 1
6. TBP and TAFIs
RNA Pol I promoters
1. Generally consists of a bipartite
sequence in the region preceding the
start site, including core element and
the upstream control elements (UCE).
2. 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.
RNA Pol I promoters in human cells
Two ancillary factors (UBF & SL1)
of RNA Pol I & their roles in
transcription initiation
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.
Selectivity factor 1 (SL1)
1. Does not bind to promoters by itself
2. Binds to and stabilizes the UBF-DNA
complex.
3. Interacts with the free downstream
part of the core element.
4. Recruit RNA Pol I to bind and to
initiate the transcription.
Subunits of SL1
SL1 consists of 4 proteins.
1. 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.
2. 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
The initiation complex
UBF
UBF
TAFIs
proposed in your text book
TAFIs
TBP RNA
Pol I
TAFIs
It is not known which representation one is
more accurate.
Other rRNA genes (simple)
In a simple eukaryote,
Acanthamoeba(棘阿米巴属 ), 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.
5. 7 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
5.7-1. 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
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).
5.7-2. 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).
• 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
5.7-3 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
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.
TFIIIA
TFIIIC
TFIIIB
Pol III
5.7-4 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
1. 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
5.7-5 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’
5.8 RNA Pol II genes:
promoters and enhancers
1. RNA Pol II
2.Cis-acting elements
• Promoters
• Upstream regulatory elements
• Enhancers
enhancer
Initiation element
TATA
Inr
Upstream element
mRNA
Down-stream element
5.8-1 RNA Pol II
1. located in nucleoplasm
2. catalyzing the synthesis of the
mRNA precursors for all proteincoding genes.
3. RNA Pol Ⅱ-transcribed premRNAs are processed through
cap addition, poly(A) tail addition
and splicing.
5.8-2 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.
转录起始点
• 转录起始位点没有广泛的序列同源性，但第一个
碱基为腺嘌呤，而两侧是嘧啶碱基。这个区域被
称为起始子（initiator， Inr），序列可表示为
PyPyANT/APyPy。Inr元件位于-2～+4。仅由Inr
元件组成的启动子是具有可被RNA聚合酶II识别的
最简单启动子形式
• 转录起始位点与TATA box一起组成核心启动子；
• 上游调控元件具有促进转录作用
转录起始点与核心启动子
( class II recognized by RNA polymerase II )
l
Cap site ; initiation point (+1)
Capping m7GpppA/G------70 ±---AUG---(in mRNA)
起始位点区 PyPyANT/(A)PyPy
1-4Kb
-70
-30
GC island CAAC/T box
Enhancer
TATA box
+1
Cap
UPE
core promoter
Promoter (basic factor)
•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.
5.8-3 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 100200 bp from the promoter, and plays
an important role in ensuring efficient
transcription.
5.8-4 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
5.9 General transcription
factors and RNA PolⅡ initiation
1. RNA Pol II basal transcription factors
2. 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
1. TFIID:
Multiprotein
Complex,
including TBP,
other proteins
are known as
TAFIIs.
TBP is the only
protein binds to
TATA box
TBP:
1. a general
transcription
factor bound
to DNA at the
TATA box.
2. a general
transcription
required by
all 3 RNA pol.
TBP:
3. Has a saddle structure with an overall dyad
symmetry.
Outer surface (with ?)
TBP
DNA
Inner surface (with ?)
TBP
45o Kink
TBP causes DNA bending
2. TFIIA
• binds to
TFIID
• stabilizes
TFIID-DNA
complex
• contains at
least 3
subunits
3. TFIIB &
RNA Pol
binding
• binds to
TFIID
•Binds to RNA
Pol with TFIIF
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.
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.