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
The revised central dogma2008
RNA processing
Gene regulation
Ch 6,The structures of DNA and RNA
Ch 8,The replication of DNA基因组的保持 基因组的表达
4
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 the
transcription cycle
2,The transcription cycle in
bacteria
3,Transcription in eukaryotes
4,Transcription by RNA
polymerase I and III
Molecular Biology Course
Transcription vs replication
6
Transcription is chemically
and enzymatically very
similar to DNA replication.
7
Some important
differences:
1.RNA is made of ribonucleotides
2.RNA polymerase catalyzes the
reaction,which does not require
a primer (de novo synthesis)
3.The RNA product does not remain
base-paired to the template DNA
strand
4.Less accurate (error rate,10-4)
8
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?)
9
Fig 12-1 Transcription of DNA into
RNA
Transcription bubble
10
Topic 1:
RNA Polymerase and
The Transcription Cycle
CHAPTER12,Mechanisms of Transcription
See the interactive animation
11
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 III transcribe tRNA gene,
some small nuclear RNA genes
and the 5S rRNA genes
13
Table 12-1,The subunits of
RNA polymerases
14
The bacterial RNA polymerase
The core enzyme alone synthesizes RNA
a
a
b
b’
w
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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
16
“Crab claw” shape of RNAP
(The shape of DNA pol is__)
Active center cleft
17
There are various channels allowing
DNA,RNA and ribonucleotides (rNTPs)
into and out of the enzyme’s active
center cleft
18
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)
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Transcription initiation
involves 3 defined steps
1,Forming closed complex
2,Forming open complex
3,Promoter escape,stable
ternary complex
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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
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Open complex
the DNA strand separate
over a distance of ~14 bp
(-11 to +3 ) around the
start site (+1 site)
transcription bubble forms
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Stable ternary complex
The enzyme escapes
from the promoter
The transition to the
elongation phase
Stable ternary complex
=DNA +RNA + enzyme
24
Fig 12-3-initiation
Binding (closed
complex)
Promoter,melting”
(open complex)
Initial
transcription
25
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
26
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.
27
Fig 12-3-Elongation and
termination
Termination
Elongation
28
Topic 2
The transcription
cycle in bacteria
CHAPTER12,Mechanisms of Transcription
29
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?
31
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
32
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
33BOX 12-1 Figure 1
Consensus sequence of
the -35 and -10 region
34
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.
35
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
36
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
37
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.
38
Fig 12-6,regions of?
Region 4 recognizes -35 element
Region 2 recognizes -10 element
Region 3 recognizes the extended –10
element
39
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
40
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,
41
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
42
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)
44
the opening of the DNA
double helix,called,melting”,
at positions -11 and +3.
Change of the promoter DNA
45
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
46
NTP uptake
channel is in the
back
Fig 12-8 channels into and out of the
open complex
DNA entering
channel
47
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
50
NTP uptake
channel is in the
back
Fig 12-8 channels into and out of the
open complex
DNA entering
channel
51
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
53
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
54
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 12-12
56
Weakest base
pairing,A:U
make the
dissociation easier
Fig 12-13
transcription
termination
57
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
58
Fig 12-11 the r transcription terminator
Hexamer,
Open ring
RNA tread
trough the
“ring”
59
Topic 3:
transcription in
eukaryotes
CHAPTER12,Mechanisms of Transcription
60
A comparison with
bacterial transcription
61
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
62
Comparison of eukaryotic and
prokaryotic promoter recognition
Eukaryotes,general transcription
factors (GTFs) (sufficient for initiate
the transcription by RNA Pol in vitro
but not in vivo),TFI factors for RNAP
I,TFII factors for RNAP II and
TFIII factors for RNAP III
Prokaryotes,? factors
63
In addition to the RNAP and
GTFs,in vivo transcription also
requires
Mediator complex
DNA-binding regulatory
proteins
chromatin-modifying
enzymes
Why
64
1,Cis-acting elements,core promoter
& regulatory sequences
2,Formation of the pre-initiation
complex
3,Promoter escape
4,The function of each GTF (in vitro)
5,Additional proteins for in vivo
transcription.
Transcription initiation
for RNA Pol II
65
RNA polymerase II core
promoters are made up of
combinations of 4 different
sequence elements
Eukaryotic core promoter (40-60
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 elements
(DPE,DCE,MTE)
Fig 12-12,Pol II core promoter
67
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
68
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.
Promoter escape requires
phosphorylation of the
polymerase,tail”
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
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Kinase vs Phosphotase
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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
73
The other GTFs also have
specific roles in initiation
~10 TAFs in TFIID,(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) base specific interaction with the
major groove of BRE,(3)Binding to
TBP-TATA complex asymmetrically,
which accounts for the unidirectional
transcription,(4) Also contacts RNA
Pol II-a bridge.
Fig 12-17 TFIIB-TBP-promoter complex
75
TFIIF,(1) a two subunit factor,(2)
Associates and recruits RNA Pol II to the
promoter,and this binding 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
function as ATPase and one is protein
kinase,(3) important for promoter
melting and escape,(4) ATPase also
functions in nucleotide mismatch repair,
called transcription-coupled repair,
76
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 12-18 assembly of the pre-initiation
complex in presence of mediator,
nucleosome modifiers and remodelers,
and transcriptional activators
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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 12-19 comparison
of the yeast and
human mediators
80
Transcription elongation
81
Elongating polymerase
must deal with histons in
its path.
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Figure 12-22
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A new set of factors
stimulate Pol II elongation
and RNA proofreading
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A brief comparison with
bacterial polymerase elongation
83
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
Phosphoylation of the CTD leads to an
exchange of initiation factors for those
factors required for elongation and RNA
processing.
84
Various proteins are thought to
stimulate elongation by Pol II
P-TEFb (a kinase stimulating elongation in 3
separate ways),
1,Phosphorylates the serine residue at
position 2 of the CTD repeats.
2,Activates hSPT5.
3,Recruits TAT-SF1.
TFIIS,(1) Stimulates the overall rate of
elongation by resolving the polymerase
pausing,(2) Proofreading (Fig,12-21)
85
Fig 12-21 TFIIS and GreB act in
analogous way
86
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 to remove noncoding
introns (Chapter 13)
Polyadenylation (多聚腺苷化 ) of the
3’ end
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Function of 5′cap and 3’
poly(A) tail
Protection of mRNA from degradation
by exonucleases (5’-3’ or 3’-5’).
Increase translational efficiency.
5’ cap facilitates mRNA transport to
cytoplasm.
Facilitate the splicing of first intron by
5’ cap and the last intron by poly(A)
tail,
88
Fig 12-20 RNA processing
enzymes are recruited by the
tail of polymerase
Dephosphorylation of Ser5 within the CTD tail
leads to dissociation of capping machinery
Further phosphorylation of Ser2 recruits the
splicing machinery
89
Evidence,this is an overlap in
proteins involved in elongation
and those for RNA processing.
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
90
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
91
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
RNA processing 2
3’ end polyadenylation
92
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 12-24
polyadenylation
and termination
93
Transcription termination
94
Transcription termination is
linked to RNA destruction by
a highly processive RNase.
Torpedo model,The second RNA
molecule without a cap is recognized and
degrade by a possessive RNase-?Pol II
dissociation from DNA template,
Allosteric model,The transfer of the 3’
processing enzyme to RNAP II induces
conformational change? RNAP II
processivity reduces? spontaneous
termination.
Fig,12-25
96
Topic 4:
Transcription by RNA
polymerase I and III
CHAPTER12,Mechanisms of Transcription
97
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 12-26 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
99Fig 12-27 Pol III core promoter
TFIIIC binds to the promoter,recruiting
TFIIIB,which in turn recruits RNAP III
Pol III promoter are found
downstream of transcription
start site.
100
1,RNA polymerases (RNAP,真核和原核的异同 )
and transcription cycle (Initiation,
elongation and termination)
2,Transcription cycle in bacteria,
Initiation,(1) The feature of?70 promoters,(2)
Promoter binding by?70 transcription factor
(recognition mechanism),(3) Transition to open
complex,(4) Promoter escape and transition to the
ternary complex,
Elongation and editing by polymerase (10-4)
Termination,Rho-independent and Rho-
dependent mechanism.
Key points of the chapter
101
3,Transcription in eukaryotes by RNAP II,
---Initiation
(i) Cis-acting elements,core promoter & regulatory
sequences
(ii) Formation of the pre-initiation complex
(iii) Promoter escape and the CTD tail
(iv) The function of each GTF (in vitro)
(v) Additional proteins for in vivo transcription,
---Elongation and proofreading involve a new
set of GTFs (What)
---Coupled with RNA processing (How)
---5’ capping and 3’ polyadenylation
(mechanisms)
---Termination,an RNase and two models
102
4,Transcription in eukaryotes by RNAP I and
III-initiation,Promoter binding and
formation of the closed complex.
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
The revised central dogma2008
RNA processing
Gene regulation
Ch 6,The structures of DNA and RNA
Ch 8,The replication of DNA基因组的保持 基因组的表达
4
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 the
transcription cycle
2,The transcription cycle in
bacteria
3,Transcription in eukaryotes
4,Transcription by RNA
polymerase I and III
Molecular Biology Course
Transcription vs replication
6
Transcription is chemically
and enzymatically very
similar to DNA replication.
7
Some important
differences:
1.RNA is made of ribonucleotides
2.RNA polymerase catalyzes the
reaction,which does not require
a primer (de novo synthesis)
3.The RNA product does not remain
base-paired to the template DNA
strand
4.Less accurate (error rate,10-4)
8
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?)
9
Fig 12-1 Transcription of DNA into
RNA
Transcription bubble
10
Topic 1:
RNA Polymerase and
The Transcription Cycle
CHAPTER12,Mechanisms of Transcription
See the interactive animation
11
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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12
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 III transcribe tRNA gene,
some small nuclear RNA genes
and the 5S rRNA genes
13
Table 12-1,The subunits of
RNA polymerases
14
The bacterial RNA polymerase
The core enzyme alone synthesizes RNA
a
a
b
b’
w
15
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
16
“Crab claw” shape of RNAP
(The shape of DNA pol is__)
Active center cleft
17
There are various channels allowing
DNA,RNA and ribonucleotides (rNTPs)
into and out of the enzyme’s active
center cleft
18
Transcription by RNA
polymerase proceeds
in a series of steps
Initiation
Elongation
Termination
RN
A
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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)
20
Transcription initiation
involves 3 defined steps
1,Forming closed complex
2,Forming open complex
3,Promoter escape,stable
ternary complex
RN
A
po
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m
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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
22
Open complex
the DNA strand separate
over a distance of ~14 bp
(-11 to +3 ) around the
start site (+1 site)
transcription bubble forms
23
Stable ternary complex
The enzyme escapes
from the promoter
The transition to the
elongation phase
Stable ternary complex
=DNA +RNA + enzyme
24
Fig 12-3-initiation
Binding (closed
complex)
Promoter,melting”
(open complex)
Initial
transcription
25
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
26
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.
27
Fig 12-3-Elongation and
termination
Termination
Elongation
28
Topic 2
The transcription
cycle in bacteria
CHAPTER12,Mechanisms of Transcription
29
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?
31
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
32
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
33BOX 12-1 Figure 1
Consensus sequence of
the -35 and -10 region
34
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.
35
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
36
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
37
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.
38
Fig 12-6,regions of?
Region 4 recognizes -35 element
Region 2 recognizes -10 element
Region 3 recognizes the extended –10
element
39
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
40
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,
41
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
42
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)
44
the opening of the DNA
double helix,called,melting”,
at positions -11 and +3.
Change of the promoter DNA
45
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
46
NTP uptake
channel is in the
back
Fig 12-8 channels into and out of the
open complex
DNA entering
channel
47
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
50
NTP uptake
channel is in the
back
Fig 12-8 channels into and out of the
open complex
DNA entering
channel
51
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
53
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
54
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 12-12
56
Weakest base
pairing,A:U
make the
dissociation easier
Fig 12-13
transcription
termination
57
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
58
Fig 12-11 the r transcription terminator
Hexamer,
Open ring
RNA tread
trough the
“ring”
59
Topic 3:
transcription in
eukaryotes
CHAPTER12,Mechanisms of Transcription
60
A comparison with
bacterial transcription
61
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
62
Comparison of eukaryotic and
prokaryotic promoter recognition
Eukaryotes,general transcription
factors (GTFs) (sufficient for initiate
the transcription by RNA Pol in vitro
but not in vivo),TFI factors for RNAP
I,TFII factors for RNAP II and
TFIII factors for RNAP III
Prokaryotes,? factors
63
In addition to the RNAP and
GTFs,in vivo transcription also
requires
Mediator complex
DNA-binding regulatory
proteins
chromatin-modifying
enzymes
Why
64
1,Cis-acting elements,core promoter
& regulatory sequences
2,Formation of the pre-initiation
complex
3,Promoter escape
4,The function of each GTF (in vitro)
5,Additional proteins for in vivo
transcription.
Transcription initiation
for RNA Pol II
65
RNA polymerase II core
promoters are made up of
combinations of 4 different
sequence elements
Eukaryotic core promoter (40-60
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 elements
(DPE,DCE,MTE)
Fig 12-12,Pol II core promoter
67
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
68
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.
Promoter escape requires
phosphorylation of the
polymerase,tail”
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
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Kinase vs Phosphotase
71
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
73
The other GTFs also have
specific roles in initiation
~10 TAFs in TFIID,(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) base specific interaction with the
major groove of BRE,(3)Binding to
TBP-TATA complex asymmetrically,
which accounts for the unidirectional
transcription,(4) Also contacts RNA
Pol II-a bridge.
Fig 12-17 TFIIB-TBP-promoter complex
75
TFIIF,(1) a two subunit factor,(2)
Associates and recruits RNA Pol II to the
promoter,and this binding 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
function as ATPase and one is protein
kinase,(3) important for promoter
melting and escape,(4) ATPase also
functions in nucleotide mismatch repair,
called transcription-coupled repair,
76
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 12-18 assembly of the pre-initiation
complex in presence of mediator,
nucleosome modifiers and remodelers,
and transcriptional activators
78
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 12-19 comparison
of the yeast and
human mediators
80
Transcription elongation
81
Elongating polymerase
must deal with histons in
its path.
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Figure 12-22
82
A new set of factors
stimulate Pol II elongation
and RNA proofreading
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A brief comparison with
bacterial polymerase elongation
83
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
Phosphoylation of the CTD leads to an
exchange of initiation factors for those
factors required for elongation and RNA
processing.
84
Various proteins are thought to
stimulate elongation by Pol II
P-TEFb (a kinase stimulating elongation in 3
separate ways),
1,Phosphorylates the serine residue at
position 2 of the CTD repeats.
2,Activates hSPT5.
3,Recruits TAT-SF1.
TFIIS,(1) Stimulates the overall rate of
elongation by resolving the polymerase
pausing,(2) Proofreading (Fig,12-21)
85
Fig 12-21 TFIIS and GreB act in
analogous way
86
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 to remove noncoding
introns (Chapter 13)
Polyadenylation (多聚腺苷化 ) of the
3’ end
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Function of 5′cap and 3’
poly(A) tail
Protection of mRNA from degradation
by exonucleases (5’-3’ or 3’-5’).
Increase translational efficiency.
5’ cap facilitates mRNA transport to
cytoplasm.
Facilitate the splicing of first intron by
5’ cap and the last intron by poly(A)
tail,
88
Fig 12-20 RNA processing
enzymes are recruited by the
tail of polymerase
Dephosphorylation of Ser5 within the CTD tail
leads to dissociation of capping machinery
Further phosphorylation of Ser2 recruits the
splicing machinery
89
Evidence,this is an overlap in
proteins involved in elongation
and those for RNA processing.
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
90
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
91
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
RNA processing 2
3’ end polyadenylation
92
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 12-24
polyadenylation
and termination
93
Transcription termination
94
Transcription termination is
linked to RNA destruction by
a highly processive RNase.
Torpedo model,The second RNA
molecule without a cap is recognized and
degrade by a possessive RNase-?Pol II
dissociation from DNA template,
Allosteric model,The transfer of the 3’
processing enzyme to RNAP II induces
conformational change? RNAP II
processivity reduces? spontaneous
termination.
Fig,12-25
96
Topic 4:
Transcription by RNA
polymerase I and III
CHAPTER12,Mechanisms of Transcription
97
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 12-26 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
99Fig 12-27 Pol III core promoter
TFIIIC binds to the promoter,recruiting
TFIIIB,which in turn recruits RNAP III
Pol III promoter are found
downstream of transcription
start site.
100
1,RNA polymerases (RNAP,真核和原核的异同 )
and transcription cycle (Initiation,
elongation and termination)
2,Transcription cycle in bacteria,
Initiation,(1) The feature of?70 promoters,(2)
Promoter binding by?70 transcription factor
(recognition mechanism),(3) Transition to open
complex,(4) Promoter escape and transition to the
ternary complex,
Elongation and editing by polymerase (10-4)
Termination,Rho-independent and Rho-
dependent mechanism.
Key points of the chapter
101
3,Transcription in eukaryotes by RNAP II,
---Initiation
(i) Cis-acting elements,core promoter & regulatory
sequences
(ii) Formation of the pre-initiation complex
(iii) Promoter escape and the CTD tail
(iv) The function of each GTF (in vitro)
(v) Additional proteins for in vivo transcription,
---Elongation and proofreading involve a new
set of GTFs (What)
---Coupled with RNA processing (How)
---5’ capping and 3’ polyadenylation
(mechanisms)
---Termination,an RNase and two models
102
4,Transcription in eukaryotes by RNAP I and
III-initiation,Promoter binding and
formation of the closed complex.
