Welcome Each of
You to My
Molecular Biology
Class
2
Molecular Biology of the Gene,
5/E --- Watson et al,(2004)
Part I,Chemistry and Genetics
Part II,Maintenance of the Genome
Part III,Expression of the Genome
Part IV,Regulation
Part V,Methods
3
Ch 12,Mechanisms of transcription
Ch 13,RNA splicing
Ch 14,Translation
Ch 15,The genetic code
Part III,Expression of
the Genome
Chapter 12,Mechanisms
of Transcription
1,RNA polymerase and
transcription cycle
2,The transcription cycle in
bacteria
3,Transcription in eukaryotes
?Molecular Biology Course
5
The Central Dogma
DNA RNA PROTEIN
Transcription Translation
replication
6
Transcription is very similar
to DNA replication but there
are some important
differences:
1.RNA is made of ribonucleotides
2.RNA polymerase catalyzes the
reaction
3.The synthesized RNA does not
remain base-paired to the
template DNA strand
4.Less accurate (error rate,10-4)
7
5.Transcription selectively copies
only certain parts of the genome
and makes one to several
hundred,or even thousand,
copies of any given section of the
genome,(Replication?)
8
Fig 12-1 Transcription of DNA into
RNA
Transcription bubble
9
Topic 1:
RNA Polymerase and
The Transcription Cycle
CHAPTER12,Mechanisms of Transcription
10
RNA polymerases come in
different forms,but share
many features
? RNA polymerases performs
essentially the same reaction
in all cells
? Bacteria have only a single
RNA polymerases while in
eukaryotic cells there are
three,RNA Pol I,II and III
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? RNA Pol II is the focus of
eukaryotic transcription,because
it is the most studied polymerase,
and is also responsible for
transcribing most genes-indeed,
essentially all protein-encoding
genes
? RNA Pol I transcribe the large
ribosomal RNA precursor gene
? RNA Pol II transcribe tRNA gene,
some small nuclear RNA genes
and the 5S rRNA genes
12
Table 12-1,The subunits of
RNA polymerases
13
The bacterial RNA polymerase
The core enzyme alone synthesizes RNA
a
a
b
b’
w
14
a
a
b’
w
RPB3
RPB11
RPB2
RPB1
RPB6
Fig 12-2 RNAP
Comparison
The same color
indicate the
homologous of
the two enzymes
prokaryotic
eukaryotic
b
15
“Crab claw” shape of RNAP
(The shape of DNA pol is__)
Active center cleft
16
There are various channels allowing
DNA,RNA and ribonucleotides (rNTPs)
into and out of the enzyme’s active
center cleft
17
Transcription by RNA
polymerase proceeds
in a series of steps
?Initiation
?Elongation
?Termination
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Initiation
? Promoter,the DNA sequence that
initially binds the RNA polymerase
? The structure of promoter-
polymerase complex undergoes
structural changes to proceed
transcription
? DNA at the transcription site
unwinds and a ―bubble‖ forms
? Direction of RNA synthesis occurs
in a 5’-3’ direction (3’-end growing)
19
Fig 12-3-initiation
Binding (closed
complex)
Promoter ―melting‖
(open complex)
Initial
transcription
20
Elongation
? Once the RNA polymerase has
synthesized a short stretch of RNA
(~ 10 nt),transcription shifts into
the elongation phase.
? This transition requires further
conformational change in
polymerase that leads it to grip the
template more firmly.
? Functions,synthesis RNA,unwinds
the DNA in front,re-anneals it
behind,dissociates the growing
RNA chain
21
Termination
?After the polymerase
transcribes the length of the
gene (or genes),it will stop
and release the RNA transcript.
?In some cells,termination
occurs at the specific and well-
defined DNA sequences called
terminators,Some cells lack
such termination sequences.
22
Fig 12-3-Elongation and
termination
Termination
Elongation
23
Transcription initiation
involves 3 defined steps
1,Forming closed complex
2,Forming open complex
3,Promoter escape
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?The initial binding of
polymerase to a promoter
?DNA remains double
stranded
?The enzyme is bound to
one face of the helix
Closed complex
25
Open complex
?the DNA strand separate
over a distance of ~14 bp
(-11 to +3 ) around the
start site (+1 site)
?Replication bubble forms
26
Stable ternary complex
?The enzyme escapes
from the promoter
?The transition to the
elongation phase
?Stable ternary complex
=DNA +RNA + enzyme
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Topic 2
The transcription
cycle in bacteria
CHAPTER12,Mechanisms of Transcription
28
2-1 Bacterial promoters
vary in strength and
sequences,but have
certain defining features
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Figure 12-4
,
Holoenzyme=
? factor + core enzyme
In cell,RNA polymerase initiates
transcription only at promoters,
Who confers the polymerase binding
specificity?
30
? The predominant ? factor in E,
coli is ?70,
? Promoter recognized by ?70
contains two conserved
sequences (-35 and –10
regions/elements) separated by
a non-specific stretch of 17-19 nt,
? Position +1 is the transcription
start site.
Promoters recognized
by E,coli ? factor
31
Fig 12-5a,bacterial promoter
The distance is conserved
1,?70 promoters contain recognizable
–35 and –10 regions,but the
sequences are not identical,
2,Comparison of many different
promoters derives the consensus
sequences reflecting preferred –10
and –35 regions
32BOX 12-1 Figure 1
Consensus sequence of
the -35 and -10 region
33
3.Promoters with sequences closer to
the consensus are generally
―stronger‖ than those match less
well,(What does,stronger” mean?)
4.The strength of the promoter
describes how many transcripts it
initiates in a given time.
34
Fig 12-5b bacterial promoter
Confers additional specificity
UP-element is an additional DNA
elements that increases
polymerase binding by providing
the additional interaction site for
RNA polymerase
35
Fig 12-5c bacterial promoter
Another class of ?70 promoter lacks a
–35 region and has an ―extended –
10 element‖ compensating for the
absence of –35 region
36
2-2 The ? factor mediates
binding of polymerase to
the promoter
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? The ?70 factor comprises four
regions called ? region 1 to ?
region 4.
37
Fig 12-6,regions of ?
Region 4 recognizes -35 element
Region 2 recognizes -10 element
Region 3 recognizes the extended –10
element
38
Binding of –35 Two helices within
region 4 form a common DNA-binding
motif,called a helix-turn-helix motif
Fig 5-20* Helix-turn-
helix DNA-binding
motif
One helix inserts
into the DNA major
groove interacting
with the bases at the
–35 region,The
other helix lies
across the top of the
groove,contacting
the DNA backbone
39
Interaction with –10 is more
elaborate (精细 ) and less understood
? The -10 region is within DNA
melting region
? The a helix recognizing –10 can
interacts with bases on the
non-template strand to
stabilize the melted DNA,
40
UP-element is recognized by
a carboxyl terminal domain of the a-
subunit (aCTD),but not by ? factor
Fig 12-7 ? and a subunits recruit RNA
pol core enzyme to the promoter
41
2-3 Transition to the open
complex involves structural
changes in RNA polymerase
and in the promoter DNA
This transition is called
Isomerization (异构化 )
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? For ?70 –containing RNA
polymerase,isomerization is a
spontaneous conformational
change in the DNA-enzyme
complex to a more
energetically favorable form,
(No extra energy requirement)
43
?the opening of the DNA
double helix,called ―melting‖,
at positions -11 and +3.
Change of the promoter DNA
44
The striking structural
change in the polymerase
? 1,the b and b’ pincers down
tightly on the downstream DNA
? 2,A major shift occurs in the N-
terminal region of ? (region 1.1)
shifts,In the closed complex,?
region 1.1 is in the active center;
in the open complex,the region
1.1 shift to the outside of the
center,allowing DNA access to
the cleft
45
NTP uptake
channel is in the
back
Fig 12-8 channels into and out of the
open complex
DNA entering
channel
46
Transcription is initiated by
RNA polymerase without
the need for a primer
Initiation requires:
? The initiating NTP (usually an A)
is placed in the active site
? The initiating ATP is held tightly
in the correct orientation by
extensive interactions with the
holoenzyme
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RNA polymerase
synthesizes several short
RNAs before entering the
elongation phase
Abortive initiation,the
enzyme synthesizes and
releases short RNA
molecules less than 10 nt,
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Structural barrier for the
abortive initiation
? The 3.2 region of ? factor lies
in the middle of the RNA exit
channel in the open complex.
? Ejection of this region from
the channel (1) is necessary
for further RNA elongation,(2)
takes the enzyme several
attempts
49
NTP uptake
channel is in the
back
Fig 12-8 channels into and out of the
open complex
DNA entering
channel
50
The elongating polymerase
is a processive machine
that synthesizes and
proofreads RNA
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1,DNA enters the polymerase
between the pincers
2,Strand separation in the catalytic
cleft
3,NTP addition
4,RNA product spooling out (Only 8-
9 nts of the growing RNA remain
base-paired with the DNA
template at any given time)
5,DNA strand annealing in behind
Synthesizing by RNA polymerase
52
? Pyrohosphorolytic （焦磷酸键解）
editing,the enzyme catalyzes the
removal of an incorrectly
inserted ribonucleotide by
reincorporation of PPi.
? Hydrolytic （水解） editing,the
enzyme backtracks by one or
more nucleotides and removes
the error-containing sequence,
This is stimulated by Gre factor,
a elongation stimulation factor.
Proofreading by RNA polymerase
53
Transcription is terminated
by signals within the RNA
sequence
Terminators,the sequences
that trigger the elongation
polymerase to dissociate
from the DNA
? Rho-dependent (requires Rho
protein)
? Rho-independent,also called
intrinsic (内在 ) terminator
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Rho-independent terminator
contains a short inverted repeat (~20
bp) and a stretch of ~8 A:T base pairs,
Fig 12-9
55
Weakest base
pairing,A:U
make the
dissociation easier
Fig 12-10
transcription
termination
56
Rho (r) -dependent terminators
? Have less well-characterized RNA
elements,and requires Rho protein for
termination
? Rho is a ring-shaped single-stranded
RNA binding protein,like SSB
? Rho binding can wrest (夺取 ) the RNA
from the polymerase-template complex
using the energy from ATP hydrolysis
? Rho binds to rut (r utilization) RNA sites
? Rho does not bind the translating RNA
57
Fig 12-11 the r transcription terminator
Hexamer,
Open ring
RNA tread
trough the
“ring”
58
Topic 3:
transcription in
eukaryotes
CHAPTER12,Mechanisms of Transcription
59
Comparison of eukaryotic and
prokaryotic RNA polymerases
Eukaryotes,Three polymerase
transcribes different class of genes,
Pol I-large rRNA genes; Pol II-
mRNA genes; Pol III- tRNA,5S
rRNA and small nuclear RNA genes
(U6)
Prokaryotes,one polymerase
transcribes all genes
60
Comparison of eukaryotic and
prokaryotic promoter recognition
Eukaryotes,general transcription
factors (GTFs),TFI factors for
RNAP I,TFII factors for RNAP II
and TFIII factors for RNAP III
Prokaryotes,? factors
61
In addition to the RNAP and
GTFs,in vivo transcription also
requires
?Mediator complex
?DNA-binding regulatory
proteins
?chromatin-modifying
enzymes
Why
62
RNA polymerase II core
promoters are made up of
combinations of 4 different
sequence elements
Eukaryotic core promoter (~40
nt),the minimal set of sequence
elements required for accurate
transcription initiation by the Pol
II machinery in vitro
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? TFIIB recognition element (BRE)
? The TATA element/box
? Initiator (Inr)
? The downstream promoter element
(DPE)
Fig 12-12,Pol II core promoter
64
The sequence elements other than
the core promoter that are required
to regulate the transcription
efficiency
Those increasing transcription:
? Promoter proximal elements
? Upstream activator sequences
(UASs)
? Enhancers
Those repressing elements,silencers,
boundary elements,insulators (绝缘
体 )
Regulatory sequences
65
RNA Pol II forms a pre-
initiation complex with
GTFs at the promoter
The involved GTFIIs (general
transcription factor for Pol II)
?TFIID=TBP (TATA box
binding protein) + TAFs
(TBP association factors)
?TFIIA,B,F,E,H
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1,TBP in TFIID binds to the
TATA box
2,TFIIA and TFIIB are
recruited with TFIIB
binding to the BRE
3,RNA Pol II-TFIIF complex
is then recruited
4,TFIIE and TFIIH then bind
upstream of Pol II to form
the pre-initiation complex
5,Promoter melting using
energy from ATP
hydrolysis by TFIIH )
6,Promoter escapes after
the phosphorylation of the
CTD tail
67
Promoter escape
? Stimulated by phosphorylation of
the CTD (C-terminal domain) tail
of the RNAP II
? CTD contains the heptapeptide
repeat Tyr-Ser-Pro-Thr-Ser-Pro-Ser
? Phosphorylation of the CTD ―tail‖ is
conducted by a number of specific
kinases including a subunit of TFIIH
68
TBP binds to and distorts
DNA using a b sheet
inserted into the minor
groove
? Unusual
(P367 for the
detailed
mechanism)
? The need for
that protein
to distort the
local DNA
structure
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?A:T base pairs (TATA box)
are favored because they
are more readily distorted
to allow initial opening of
the minor groove
70
The other GTFs also have
specific roles in initiation
? ~ 10 TAFs,(1) two of them
bind DNA elements at the
promoter (Inr and DPE); (2)
several are histone-like TAFs
and might bind to DNA similar
to that histone does; (3) one
regulates the binding of TBP to
DNA
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? TFIIB,(1) a single polypeptide chain,
(2) asymmetric binding to TBP and the
promoter DNA (BRE),(3)bridging TBP
and the polymerase,(4) the N-terminal
inserting in the RNA exit channel
resembles the ?3.2,
Fig 12-13 TFIIB-TBP-promoter complex
72
? TFIIF,(1) a two subunit factor,(2)
binding of Pol II-TFIIF stabilizes the
DNA-TBP-TFIIB complex,which is
required for the followed factor binding
? TFIIE,recruits and regulates TFIIH
? TFIIH,(1) controls the ATP-dependent
transition of the pre-initiation complex
to the open complex,(2) contains 9
subunits and is the largest GTF; two
functions as ATPase and one is protein
kinase,(3) important for promoter
melting and escape,(4) ATPase
functions in nucleotide mismatch repair,
called transcription-coupled repair,
73
in vivo,transcription
initiation requires
additional proteins
? The mediator complex
? Transcriptional regulatory
proteins
? Nucleosome-modifying enzymes
To counter the real situation that
the DNA template in vivo is
packed into nucleosome and
chromatin
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Fig 12-16 assembly of the pre-initiation
complex in presence of mediator,
nucleosome modifiers and remodelers,
and transcriptional activators
75
Mediator consists of
many subunits,some
conserved from yeast to
human
? More than 20 subunits
? 7 subunits show significant sequence
homology between yeast and human
? Only subunit Srb4 is essential for
transcription of essentially all Pol II
genes in vivo
? Organized in modules (模块 )
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Fig 12-17 comparison
of the yeast and
human mediators
77
Eukaryotic RNA Pol II holoenzyme
is a putative preformed complex:
Pol II + mediator + some of GTFs
Prokaryotic RNA Polymerase
holoenzyme:
core polymerase + ? factor
78
A new set of factors
stimulate Pol II
elongation and RNA
proofreading
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Transition from the initiation to
elongation involves the Pol II
enzyme shedding (摆脱 ) most of its
initiation factors (GTF and mediators)
and recruiting other factors,
(1) Elongation factors,factors that
stimulate elongation,such as TFIIS
and hSPT5.
(2) RNA processing (RNA 加工 ) factors
Recruited to the C-terminal tail of the
CTD of RNAP II to phosphorylate the
tail for elongation stimulation,
proofreading,and RNA processing
like splicing and polyadenylation.
80
Fig 12-18 RNA processing enzymes are
recruited by the tail of polymerase
81
Some elongation factors
? P-TEFb,
? phosphorylates CTD
? Activates hSPT5
? Activates TAT-SF1
? TFIIS:
? Stimulates the overall rate of
elongation by resolving the
polymerase pausing
? Proofreading
82
Elongation polymerase is
associated with a new
set of protein factors
required for various
types of RNA processing
RNA processing,
? Capping of the 5’ end of the RNA
? Splicing of the introns (most
complicated)
? Poly adenylation (多聚腺苷化 ) of the
3’ end
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Evidence,this is an overlap in
proteins involving in those events
? The elongation factor hSPT5 also
recruits and stimulates the 5’
capping enzyme
? The elongation factor TAT-SF1
recruits components for splicing
Elongation,termination of
transcription,and RNA
processing are interconnected/
coupled (偶联的 ) to ensure the
coordination (协同性 ) of these
events
84
Function of poly(A) tail
? Increased mRNA stability
? Increased translational
efficiency
? Splicing of last intron
85
Function of 5′cap
? Protection from degradation
? Increased translational
efficiency
? Transport to cytoplasm
? Splicing of first intron
86
RNA processing 1
5’ end capping
? The ―cap‖,a
methylated guanine
joined to the RNA
transcript by a 5’-5’
linkage
? The linkage
contains 3
phosphates
? 3 sequential
enzymatic reactions
? Occurs early
87
Splicing,joining the
protein coding sequences
? Dephosphorylation of Ser5 within
the CTD tail leads to dissociation of
capping machinery
? Further phosphorylation of Ser2
recruits the splicing machinery
88
3’ end polyadenylation
? Linked with the termination of
transcription
? The CTD tail is involved in
recruiting the polyadenylation
enzymes
? The transcribed poly-A signal
triggers the reactions
1,Cleavage of the message
2,Addition of poly-A
3,Termination of transcription
89
1,CPSF (cleavage and
polyadenylation specificity
factor) & CstF (cleavage
stimulation factor) bind to
the poly-A signal,leading to
the RNA cleavage
2,Poly-A polymerase (PAP)
adds ~ 200 As at the 3’ end
of the RNA,using ATP as a
substrate
Fig 12-20
polyadenylation
and termination
90
?What terminates
transcription by polymerase?
91
Models to explain the link
between polyadenylation and
termination (see the animation on
your CD)
? Model 1,The transfer of the 3’
processing enzyme to RNAP II
induces conformational change—
RNAP II processivity reduces—
spontaneous termination
? Model 2,absence of a 5’cap on the
second RNA molecule—recognized
by the RNAP II as improper—
terminate transcription
92
RNA Pol I & III recognize
distinct promoters,using
distinct sets of
transcription factors,but
still require TBP
? Pol I,transcribes rRNA precursor
encoding gene (multi-copy gene)
? Pol III,transcribes tRNA genes,
snRNA genes and 5S rRNA genes
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Pol I promoter recognition
Fig 12-21 Pol I promoter region
Upstream control element
UBF binds to the upstream of UCE,bring SL1 to the
downstream part of UCE,SL1 in turn recruits RNAP
I to the core promoter for transcription
94Fig 12-22 Pol III core promoter
TFIIIC binds to the promoter,recruiting
TFIIIB,which in turn recruits RNAP III
Pol III promoter recognition
1,Different forms,2,locates
downstream of the transcription site
95
1,RNA polymerases (RNAP,真核和原核的异同 )
and transcription cycle (Initiation is more
complicate,details in bacteria)
2,Transcription cycle in bacteria,
(1) promoters (elements),? factor (4 domains),
aCTD,abortive initiation (why?)
(2) Structures accounting for formation of the
closed complex,transitions to open complex
and then stable ternary complex.
(3) Elongation and editing by polymerase (10-4)
(4) Termination,Rho-independent and Rho-
dependent mechanism
Key points of the chapter
96
3,Transcription cycle in eukaryotes,
(1)Promoters (elements),general transcription
factors (GTF),
(2)RNAP II transcription
---the roles of GTFs and the CTD tail of RNAP II
in promoter recognition,formation of the
pre-initiation complex,promoter melting,
promoter escape
---in vivo requires mediator complex,
nucleosome modifying enzymes and
transcription regulatory proteins.
---elongation and proofreading involve a new
set of GTFs (What)
---coupled with RNA processing (How)
97
(3)RNAP I and III transcription
---GTFs and promoter recognition,formation of
the initiation complex
98
See the animations
Complete all the
excises on your study
CD,Enjoy it
Homework
You to My
Molecular Biology
Class
2
Molecular Biology of the Gene,
5/E --- Watson et al,(2004)
Part I,Chemistry and Genetics
Part II,Maintenance of the Genome
Part III,Expression of the Genome
Part IV,Regulation
Part V,Methods
3
Ch 12,Mechanisms of transcription
Ch 13,RNA splicing
Ch 14,Translation
Ch 15,The genetic code
Part III,Expression of
the Genome
Chapter 12,Mechanisms
of Transcription
1,RNA polymerase and
transcription cycle
2,The transcription cycle in
bacteria
3,Transcription in eukaryotes
?Molecular Biology Course
5
The Central Dogma
DNA RNA PROTEIN
Transcription Translation
replication
6
Transcription is very similar
to DNA replication but there
are some important
differences:
1.RNA is made of ribonucleotides
2.RNA polymerase catalyzes the
reaction
3.The synthesized RNA does not
remain base-paired to the
template DNA strand
4.Less accurate (error rate,10-4)
7
5.Transcription selectively copies
only certain parts of the genome
and makes one to several
hundred,or even thousand,
copies of any given section of the
genome,(Replication?)
8
Fig 12-1 Transcription of DNA into
RNA
Transcription bubble
9
Topic 1:
RNA Polymerase and
The Transcription Cycle
CHAPTER12,Mechanisms of Transcription
10
RNA polymerases come in
different forms,but share
many features
? RNA polymerases performs
essentially the same reaction
in all cells
? Bacteria have only a single
RNA polymerases while in
eukaryotic cells there are
three,RNA Pol I,II and III
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? RNA Pol II is the focus of
eukaryotic transcription,because
it is the most studied polymerase,
and is also responsible for
transcribing most genes-indeed,
essentially all protein-encoding
genes
? RNA Pol I transcribe the large
ribosomal RNA precursor gene
? RNA Pol II transcribe tRNA gene,
some small nuclear RNA genes
and the 5S rRNA genes
12
Table 12-1,The subunits of
RNA polymerases
13
The bacterial RNA polymerase
The core enzyme alone synthesizes RNA
a
a
b
b’
w
14
a
a
b’
w
RPB3
RPB11
RPB2
RPB1
RPB6
Fig 12-2 RNAP
Comparison
The same color
indicate the
homologous of
the two enzymes
prokaryotic
eukaryotic
b
15
“Crab claw” shape of RNAP
(The shape of DNA pol is__)
Active center cleft
16
There are various channels allowing
DNA,RNA and ribonucleotides (rNTPs)
into and out of the enzyme’s active
center cleft
17
Transcription by RNA
polymerase proceeds
in a series of steps
?Initiation
?Elongation
?Termination
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Initiation
? Promoter,the DNA sequence that
initially binds the RNA polymerase
? The structure of promoter-
polymerase complex undergoes
structural changes to proceed
transcription
? DNA at the transcription site
unwinds and a ―bubble‖ forms
? Direction of RNA synthesis occurs
in a 5’-3’ direction (3’-end growing)
19
Fig 12-3-initiation
Binding (closed
complex)
Promoter ―melting‖
(open complex)
Initial
transcription
20
Elongation
? Once the RNA polymerase has
synthesized a short stretch of RNA
(~ 10 nt),transcription shifts into
the elongation phase.
? This transition requires further
conformational change in
polymerase that leads it to grip the
template more firmly.
? Functions,synthesis RNA,unwinds
the DNA in front,re-anneals it
behind,dissociates the growing
RNA chain
21
Termination
?After the polymerase
transcribes the length of the
gene (or genes),it will stop
and release the RNA transcript.
?In some cells,termination
occurs at the specific and well-
defined DNA sequences called
terminators,Some cells lack
such termination sequences.
22
Fig 12-3-Elongation and
termination
Termination
Elongation
23
Transcription initiation
involves 3 defined steps
1,Forming closed complex
2,Forming open complex
3,Promoter escape
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?The initial binding of
polymerase to a promoter
?DNA remains double
stranded
?The enzyme is bound to
one face of the helix
Closed complex
25
Open complex
?the DNA strand separate
over a distance of ~14 bp
(-11 to +3 ) around the
start site (+1 site)
?Replication bubble forms
26
Stable ternary complex
?The enzyme escapes
from the promoter
?The transition to the
elongation phase
?Stable ternary complex
=DNA +RNA + enzyme
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Topic 2
The transcription
cycle in bacteria
CHAPTER12,Mechanisms of Transcription
28
2-1 Bacterial promoters
vary in strength and
sequences,but have
certain defining features
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Figure 12-4
,
Holoenzyme=
? factor + core enzyme
In cell,RNA polymerase initiates
transcription only at promoters,
Who confers the polymerase binding
specificity?
30
? The predominant ? factor in E,
coli is ?70,
? Promoter recognized by ?70
contains two conserved
sequences (-35 and –10
regions/elements) separated by
a non-specific stretch of 17-19 nt,
? Position +1 is the transcription
start site.
Promoters recognized
by E,coli ? factor
31
Fig 12-5a,bacterial promoter
The distance is conserved
1,?70 promoters contain recognizable
–35 and –10 regions,but the
sequences are not identical,
2,Comparison of many different
promoters derives the consensus
sequences reflecting preferred –10
and –35 regions
32BOX 12-1 Figure 1
Consensus sequence of
the -35 and -10 region
33
3.Promoters with sequences closer to
the consensus are generally
―stronger‖ than those match less
well,(What does,stronger” mean?)
4.The strength of the promoter
describes how many transcripts it
initiates in a given time.
34
Fig 12-5b bacterial promoter
Confers additional specificity
UP-element is an additional DNA
elements that increases
polymerase binding by providing
the additional interaction site for
RNA polymerase
35
Fig 12-5c bacterial promoter
Another class of ?70 promoter lacks a
–35 region and has an ―extended –
10 element‖ compensating for the
absence of –35 region
36
2-2 The ? factor mediates
binding of polymerase to
the promoter
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? The ?70 factor comprises four
regions called ? region 1 to ?
region 4.
37
Fig 12-6,regions of ?
Region 4 recognizes -35 element
Region 2 recognizes -10 element
Region 3 recognizes the extended –10
element
38
Binding of –35 Two helices within
region 4 form a common DNA-binding
motif,called a helix-turn-helix motif
Fig 5-20* Helix-turn-
helix DNA-binding
motif
One helix inserts
into the DNA major
groove interacting
with the bases at the
–35 region,The
other helix lies
across the top of the
groove,contacting
the DNA backbone
39
Interaction with –10 is more
elaborate (精细 ) and less understood
? The -10 region is within DNA
melting region
? The a helix recognizing –10 can
interacts with bases on the
non-template strand to
stabilize the melted DNA,
40
UP-element is recognized by
a carboxyl terminal domain of the a-
subunit (aCTD),but not by ? factor
Fig 12-7 ? and a subunits recruit RNA
pol core enzyme to the promoter
41
2-3 Transition to the open
complex involves structural
changes in RNA polymerase
and in the promoter DNA
This transition is called
Isomerization (异构化 )
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? For ?70 –containing RNA
polymerase,isomerization is a
spontaneous conformational
change in the DNA-enzyme
complex to a more
energetically favorable form,
(No extra energy requirement)
43
?the opening of the DNA
double helix,called ―melting‖,
at positions -11 and +3.
Change of the promoter DNA
44
The striking structural
change in the polymerase
? 1,the b and b’ pincers down
tightly on the downstream DNA
? 2,A major shift occurs in the N-
terminal region of ? (region 1.1)
shifts,In the closed complex,?
region 1.1 is in the active center;
in the open complex,the region
1.1 shift to the outside of the
center,allowing DNA access to
the cleft
45
NTP uptake
channel is in the
back
Fig 12-8 channels into and out of the
open complex
DNA entering
channel
46
Transcription is initiated by
RNA polymerase without
the need for a primer
Initiation requires:
? The initiating NTP (usually an A)
is placed in the active site
? The initiating ATP is held tightly
in the correct orientation by
extensive interactions with the
holoenzyme
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RNA polymerase
synthesizes several short
RNAs before entering the
elongation phase
Abortive initiation,the
enzyme synthesizes and
releases short RNA
molecules less than 10 nt,
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Structural barrier for the
abortive initiation
? The 3.2 region of ? factor lies
in the middle of the RNA exit
channel in the open complex.
? Ejection of this region from
the channel (1) is necessary
for further RNA elongation,(2)
takes the enzyme several
attempts
49
NTP uptake
channel is in the
back
Fig 12-8 channels into and out of the
open complex
DNA entering
channel
50
The elongating polymerase
is a processive machine
that synthesizes and
proofreads RNA
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1,DNA enters the polymerase
between the pincers
2,Strand separation in the catalytic
cleft
3,NTP addition
4,RNA product spooling out (Only 8-
9 nts of the growing RNA remain
base-paired with the DNA
template at any given time)
5,DNA strand annealing in behind
Synthesizing by RNA polymerase
52
? Pyrohosphorolytic （焦磷酸键解）
editing,the enzyme catalyzes the
removal of an incorrectly
inserted ribonucleotide by
reincorporation of PPi.
? Hydrolytic （水解） editing,the
enzyme backtracks by one or
more nucleotides and removes
the error-containing sequence,
This is stimulated by Gre factor,
a elongation stimulation factor.
Proofreading by RNA polymerase
53
Transcription is terminated
by signals within the RNA
sequence
Terminators,the sequences
that trigger the elongation
polymerase to dissociate
from the DNA
? Rho-dependent (requires Rho
protein)
? Rho-independent,also called
intrinsic (内在 ) terminator
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Rho-independent terminator
contains a short inverted repeat (~20
bp) and a stretch of ~8 A:T base pairs,
Fig 12-9
55
Weakest base
pairing,A:U
make the
dissociation easier
Fig 12-10
transcription
termination
56
Rho (r) -dependent terminators
? Have less well-characterized RNA
elements,and requires Rho protein for
termination
? Rho is a ring-shaped single-stranded
RNA binding protein,like SSB
? Rho binding can wrest (夺取 ) the RNA
from the polymerase-template complex
using the energy from ATP hydrolysis
? Rho binds to rut (r utilization) RNA sites
? Rho does not bind the translating RNA
57
Fig 12-11 the r transcription terminator
Hexamer,
Open ring
RNA tread
trough the
“ring”
58
Topic 3:
transcription in
eukaryotes
CHAPTER12,Mechanisms of Transcription
59
Comparison of eukaryotic and
prokaryotic RNA polymerases
Eukaryotes,Three polymerase
transcribes different class of genes,
Pol I-large rRNA genes; Pol II-
mRNA genes; Pol III- tRNA,5S
rRNA and small nuclear RNA genes
(U6)
Prokaryotes,one polymerase
transcribes all genes
60
Comparison of eukaryotic and
prokaryotic promoter recognition
Eukaryotes,general transcription
factors (GTFs),TFI factors for
RNAP I,TFII factors for RNAP II
and TFIII factors for RNAP III
Prokaryotes,? factors
61
In addition to the RNAP and
GTFs,in vivo transcription also
requires
?Mediator complex
?DNA-binding regulatory
proteins
?chromatin-modifying
enzymes
Why
62
RNA polymerase II core
promoters are made up of
combinations of 4 different
sequence elements
Eukaryotic core promoter (~40
nt),the minimal set of sequence
elements required for accurate
transcription initiation by the Pol
II machinery in vitro
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? TFIIB recognition element (BRE)
? The TATA element/box
? Initiator (Inr)
? The downstream promoter element
(DPE)
Fig 12-12,Pol II core promoter
64
The sequence elements other than
the core promoter that are required
to regulate the transcription
efficiency
Those increasing transcription:
? Promoter proximal elements
? Upstream activator sequences
(UASs)
? Enhancers
Those repressing elements,silencers,
boundary elements,insulators (绝缘
体 )
Regulatory sequences
65
RNA Pol II forms a pre-
initiation complex with
GTFs at the promoter
The involved GTFIIs (general
transcription factor for Pol II)
?TFIID=TBP (TATA box
binding protein) + TAFs
(TBP association factors)
?TFIIA,B,F,E,H
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1,TBP in TFIID binds to the
TATA box
2,TFIIA and TFIIB are
recruited with TFIIB
binding to the BRE
3,RNA Pol II-TFIIF complex
is then recruited
4,TFIIE and TFIIH then bind
upstream of Pol II to form
the pre-initiation complex
5,Promoter melting using
energy from ATP
hydrolysis by TFIIH )
6,Promoter escapes after
the phosphorylation of the
CTD tail
67
Promoter escape
? Stimulated by phosphorylation of
the CTD (C-terminal domain) tail
of the RNAP II
? CTD contains the heptapeptide
repeat Tyr-Ser-Pro-Thr-Ser-Pro-Ser
? Phosphorylation of the CTD ―tail‖ is
conducted by a number of specific
kinases including a subunit of TFIIH
68
TBP binds to and distorts
DNA using a b sheet
inserted into the minor
groove
? Unusual
(P367 for the
detailed
mechanism)
? The need for
that protein
to distort the
local DNA
structure
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?A:T base pairs (TATA box)
are favored because they
are more readily distorted
to allow initial opening of
the minor groove
70
The other GTFs also have
specific roles in initiation
? ~ 10 TAFs,(1) two of them
bind DNA elements at the
promoter (Inr and DPE); (2)
several are histone-like TAFs
and might bind to DNA similar
to that histone does; (3) one
regulates the binding of TBP to
DNA
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? TFIIB,(1) a single polypeptide chain,
(2) asymmetric binding to TBP and the
promoter DNA (BRE),(3)bridging TBP
and the polymerase,(4) the N-terminal
inserting in the RNA exit channel
resembles the ?3.2,
Fig 12-13 TFIIB-TBP-promoter complex
72
? TFIIF,(1) a two subunit factor,(2)
binding of Pol II-TFIIF stabilizes the
DNA-TBP-TFIIB complex,which is
required for the followed factor binding
? TFIIE,recruits and regulates TFIIH
? TFIIH,(1) controls the ATP-dependent
transition of the pre-initiation complex
to the open complex,(2) contains 9
subunits and is the largest GTF; two
functions as ATPase and one is protein
kinase,(3) important for promoter
melting and escape,(4) ATPase
functions in nucleotide mismatch repair,
called transcription-coupled repair,
73
in vivo,transcription
initiation requires
additional proteins
? The mediator complex
? Transcriptional regulatory
proteins
? Nucleosome-modifying enzymes
To counter the real situation that
the DNA template in vivo is
packed into nucleosome and
chromatin
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Fig 12-16 assembly of the pre-initiation
complex in presence of mediator,
nucleosome modifiers and remodelers,
and transcriptional activators
75
Mediator consists of
many subunits,some
conserved from yeast to
human
? More than 20 subunits
? 7 subunits show significant sequence
homology between yeast and human
? Only subunit Srb4 is essential for
transcription of essentially all Pol II
genes in vivo
? Organized in modules (模块 )
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Fig 12-17 comparison
of the yeast and
human mediators
77
Eukaryotic RNA Pol II holoenzyme
is a putative preformed complex:
Pol II + mediator + some of GTFs
Prokaryotic RNA Polymerase
holoenzyme:
core polymerase + ? factor
78
A new set of factors
stimulate Pol II
elongation and RNA
proofreading
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Transition from the initiation to
elongation involves the Pol II
enzyme shedding (摆脱 ) most of its
initiation factors (GTF and mediators)
and recruiting other factors,
(1) Elongation factors,factors that
stimulate elongation,such as TFIIS
and hSPT5.
(2) RNA processing (RNA 加工 ) factors
Recruited to the C-terminal tail of the
CTD of RNAP II to phosphorylate the
tail for elongation stimulation,
proofreading,and RNA processing
like splicing and polyadenylation.
80
Fig 12-18 RNA processing enzymes are
recruited by the tail of polymerase
81
Some elongation factors
? P-TEFb,
? phosphorylates CTD
? Activates hSPT5
? Activates TAT-SF1
? TFIIS:
? Stimulates the overall rate of
elongation by resolving the
polymerase pausing
? Proofreading
82
Elongation polymerase is
associated with a new
set of protein factors
required for various
types of RNA processing
RNA processing,
? Capping of the 5’ end of the RNA
? Splicing of the introns (most
complicated)
? Poly adenylation (多聚腺苷化 ) of the
3’ end
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Evidence,this is an overlap in
proteins involving in those events
? The elongation factor hSPT5 also
recruits and stimulates the 5’
capping enzyme
? The elongation factor TAT-SF1
recruits components for splicing
Elongation,termination of
transcription,and RNA
processing are interconnected/
coupled (偶联的 ) to ensure the
coordination (协同性 ) of these
events
84
Function of poly(A) tail
? Increased mRNA stability
? Increased translational
efficiency
? Splicing of last intron
85
Function of 5′cap
? Protection from degradation
? Increased translational
efficiency
? Transport to cytoplasm
? Splicing of first intron
86
RNA processing 1
5’ end capping
? The ―cap‖,a
methylated guanine
joined to the RNA
transcript by a 5’-5’
linkage
? The linkage
contains 3
phosphates
? 3 sequential
enzymatic reactions
? Occurs early
87
Splicing,joining the
protein coding sequences
? Dephosphorylation of Ser5 within
the CTD tail leads to dissociation of
capping machinery
? Further phosphorylation of Ser2
recruits the splicing machinery
88
3’ end polyadenylation
? Linked with the termination of
transcription
? The CTD tail is involved in
recruiting the polyadenylation
enzymes
? The transcribed poly-A signal
triggers the reactions
1,Cleavage of the message
2,Addition of poly-A
3,Termination of transcription
89
1,CPSF (cleavage and
polyadenylation specificity
factor) & CstF (cleavage
stimulation factor) bind to
the poly-A signal,leading to
the RNA cleavage
2,Poly-A polymerase (PAP)
adds ~ 200 As at the 3’ end
of the RNA,using ATP as a
substrate
Fig 12-20
polyadenylation
and termination
90
?What terminates
transcription by polymerase?
91
Models to explain the link
between polyadenylation and
termination (see the animation on
your CD)
? Model 1,The transfer of the 3’
processing enzyme to RNAP II
induces conformational change—
RNAP II processivity reduces—
spontaneous termination
? Model 2,absence of a 5’cap on the
second RNA molecule—recognized
by the RNAP II as improper—
terminate transcription
92
RNA Pol I & III recognize
distinct promoters,using
distinct sets of
transcription factors,but
still require TBP
? Pol I,transcribes rRNA precursor
encoding gene (multi-copy gene)
? Pol III,transcribes tRNA genes,
snRNA genes and 5S rRNA genes
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Pol I promoter recognition
Fig 12-21 Pol I promoter region
Upstream control element
UBF binds to the upstream of UCE,bring SL1 to the
downstream part of UCE,SL1 in turn recruits RNAP
I to the core promoter for transcription
94Fig 12-22 Pol III core promoter
TFIIIC binds to the promoter,recruiting
TFIIIB,which in turn recruits RNAP III
Pol III promoter recognition
1,Different forms,2,locates
downstream of the transcription site
95
1,RNA polymerases (RNAP,真核和原核的异同 )
and transcription cycle (Initiation is more
complicate,details in bacteria)
2,Transcription cycle in bacteria,
(1) promoters (elements),? factor (4 domains),
aCTD,abortive initiation (why?)
(2) Structures accounting for formation of the
closed complex,transitions to open complex
and then stable ternary complex.
(3) Elongation and editing by polymerase (10-4)
(4) Termination,Rho-independent and Rho-
dependent mechanism
Key points of the chapter
96
3,Transcription cycle in eukaryotes,
(1)Promoters (elements),general transcription
factors (GTF),
(2)RNAP II transcription
---the roles of GTFs and the CTD tail of RNAP II
in promoter recognition,formation of the
pre-initiation complex,promoter melting,
promoter escape
---in vivo requires mediator complex,
nucleosome modifying enzymes and
transcription regulatory proteins.
---elongation and proofreading involve a new
set of GTFs (What)
---coupled with RNA processing (How)
97
(3)RNAP I and III transcription
---GTFs and promoter recognition,formation of
the initiation complex
98
See the animations
Complete all the
excises on your study
CD,Enjoy it
Homework
