Welcome Each

of You to My
Molecular
Biology Class
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

2
 Part
Part III:
III: Expression
Expression of
of
the
the Genome
Genome
Ch 12: Mechanisms of transcription
Ch 13: RNA splicing
Ch 14: Translation
Ch 15: The genetic code

3
•Molecular Biology
Chapter 12:
Course

Mechanisms of
Transcription
1. RNA polymerase and
transcription cycle
2. The transcription cycle in
bacteria
3. Transcription in eukaryotes
The Central Dogma

Transcription Translation
DNA RNA PROTEIN
replication

5
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 )
6
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?)

7
 Transcription bubble

Fig 12-1 Transcription of DNA into

RNA

8
Transcription

Topic 1:

RNA Polymerase and

The Transcription Cycle

See the interactive animation

9
RNA polymerase and the transcription cycle

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

10
 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
11
Table 12-1: The subunits of
RNA polymerases

12
The bacterial RNA polymerase
The core enzyme alone synthesizes RNA

β
α
’
β

α
ω 13
prokaryotic β Fig 12-2 RNAP
’ Comparison
α
β

α The same color

indicate the
ω
homologous of
the two enzymes
eukaryotic RPB2

RPB3

RPB1
RPB11

RPB6 14
“Crab claw” shape of RNAP
(The shape of DNA pol is__)

Active center cleft

15
There are various channels allowing
DNA, RNA and ribonucleotides
(rNTPs) into and out of the enzyme’s
active center cleft

16
RNA polymerase and the transcription cycle

Transcription by RNA
polymerase proceeds
in a series of steps

 Initiation
 Elongation
 Termination

17
 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)

18
Fig 12-3-initiation

Binding (closed
complex)

Promoter
“melting” (open
complex)

Initial
transcription
19
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 20
 Termination
 Afterthe 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.
21
Fig 12-3-Elongation and
termination

Elongation

Termination

22
RNA polymerase and the transcription cycle

Transcription initiation
involves 3 defined steps

1. Forming closed complex

2. Forming open complex
3. Promoter escape

23
Closed complex
 The initial binding of
polymerase to a promoter
 DNA remains double
stranded
 The enzyme is bound to
one face of the helix

24
 Open complex
 theDNA strand separate
over a distance of ~14 bp
(-11 to +3 ) around the
start site (+1 site)

 Replication bubble forms

25
RNA polymerase and the transcription cycle

Stable ternary complex

 The enzyme escapes
from the promoter
 The transition to the
elongation phase
 Stable ternary complex

=DNA +RNA + enzyme

26
Transcription

Topic 2

The transcription
cycle in bacteria

27
The transcription cycle in bacteria

2-1 Bacterial promoters

vary in strength and
sequences, but have
certain defining features

28
Holoenzyme=
σ factor + core
enzyme

Figure 12-4

In cell, RNA polymerase initiates

transcription only at promoters.
Who confers the polymerase binding
specificity?
, 29
 Promoters recognized
by E. coli σ factor
 The predominant σ factor in E. coli
is σ 70 .
 Promoter recognized by σ 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.

30
 Fig 12-5a: bacterial promoter
The distance is conserved

1 . σ 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
31
 Consensus sequence of
the -35 and -10 region

BOX 12-1 Figure 1 32

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.
33
 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

34
 Fig 12-5c bacterial promoter

Another class of σ 70 promoter lacks a

–35 region and has an “extended –
10 element” compensating for the
absence of –35 region

35
 2-2 The σ factor mediates
The transcription cycle in bacteria

binding of polymerase to
the promoter
 The σ 70 factor comprises four
regions called σ region 1 to σ
region 4.

36
 Fig 12-6: regions of
σ

Region 4 recognizes -35 element

Region 2 recognizes -10 element
Region 3 recognizes the extended –10
element 37
Binding of –35 Two helices within
region 4 form a common DNA-binding
motif, called a helix-turn-helix 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

Fig 5-20* Helix-turn-

helix DNA-binding
motif 38
Interaction with –10 is more
elaborate ( 精细 ) and less understood

 The -10 region is within DNA

melting region
 The α helix recognizing –10
can interacts with bases on the
non-template strand to
stabilize the melted DNA.

39
UP-element is recognized by a
carboxyl terminal domain of the α -
subunit (α CTD), but not by σ factor

Fig 12-7 σ and α subunits recruit RNA

pol core enzyme to the promoter
40
 2-3 Transition to the open
The transcription cycle in bacteria

complex involves structural

changes in RNA polymerase
and in the promoter DNA

This transition is called

Isomerization ( 异构化 )

41
For σ 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)

42
Change of the promoter DNA

 the
opening of the DNA
double helix, called
“melting”, at positions -11
and +3.

43
The striking structural
change in the polymerase
 1. the β and β ’ pincers down
tightly on the downstream DNA
 2. A major shift occurs in the N-
terminal region of σ (region 1.1)
shifts. In the closed complex, σ
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 44
 NTP uptake
channel is in the
back

DNA entering
channel

Fig 12-8 channels into and out of the

open complex 45
 Transcription is initiated by
The transcription cycle in bacteria

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
46
 RNA polymerase
The transcription cycle in bacteria

synthesizes several short

RNAs before entering the
elongation phase

Abortive initiation: the enzyme

synthesizes and releases short
RNA molecules less than 10 nt.

47
Structural barrier for the
abortive initiation
 The 3.2 region of σ 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
48
 NTP uptake
channel is in the
back

DNA entering
channel

Fig 12-8 channels into and out of the

open complex 49
 The elongating polymerase
The transcription cycle in bacteria

is a processive machine
that synthesizes and
proofreads RNA

50
Synthesizing by RNA polymerase
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
51
Proofreading by RNA
polymerase
 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.
52
 Transcription is terminated
The transcription cycle in bacteria

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
53
Rho-independent terminator
contains a short inverted repeat (~20
bp) and a stretch of ~8 A:T base pairs.

Fig 12-9 54
 Fig 12-10
transcription
termination

Weakest base
pairing: A:U
make the
dissociation easier
55
Rho (ρ ) -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 (ρ utilization) RNA sites
 Rho does not bind the translating RNA

56
Fig 12-11 the ρ transcription terminator

RNA tread
trough the
“ring”

Hexamer,
Open ring

57
Transcription

Topic 3:
transcription in
eukaryotes

58
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
59
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: σ factors

60
In addition to the RNAP and
GTFs, in vivo transcription also
requires
 Mediator complex
 DNA-binding regulatory
proteins
 chromatin-modifying
enzymes
Why??
61
 RNA polymerase II core
The transcription in eukaryotes

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

62
Fig 12-12: Pol II core promoter

 TFIIB recognition element (BRE)

 The TATA element/box
 Initiator (Inr)
 The downstream promoter element
(DPE)
63
 Regulatory sequences
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) 64
 RNA Pol II forms a pre-
The transcription in eukaryotes

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

65
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 66
 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

67
 TBP binds to and distorts
The transcription in eukaryotes

DNA using a β sheet

inserted into the minor
groove
 Unusual
(P367 for the
detailed
mechanism)
 The need for
that protein
to distort the
local DNA
structure 68
 A:Tbase pairs (TATA box)
are favored because they
are more readily distorted
to allow initial opening of
the minor groove

69
 The other GTFs also have
The transcription in eukaryotes

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

70
  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 σ 3.2 .

Fig 12-13 TFIIB-TBP-promoter complex 71

 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.
72
 in vivo, transcription
The transcription in eukaryotes

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
73
Fig 12-16 assembly of the pre-initiation
complex in presence of mediator,
nucleosome modifiers and remodelers,
and transcriptional activators

74
 Mediator consists of
The transcription in eukaryotes

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 )
75
Fig 12-17 comparison
of the yeast and
human mediators

76
Eukaryotic RNA Pol II holoenzyme
is a putative preformed complex:
Pol II + mediator + some of GTFs

Prokaryotic RNA Polymerase

holoenzyme:
core polymerase + σ factor

77
 A new set of factors
The transcription in eukaryotes

stimulate Pol II
elongation and RNA
proofreading

78
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, 79
Fig 12-18 RNA processing enzymes are
recruited by the tail of polymerase

80
 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
81
 Elongation polymerase is
The transcription in eukaryotes

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 82
Elongation, termination of
transcription, and RNA
processing are interconnected/
coupled ( 偶联的 ) to ensure the
coordination ( 协同性 ) of these
events
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 83
 Function of poly(A) tail
 Increased mRNA stability
 Increased translational
efficiency
 Splicing of last intron

84
 Function of 5´cap
 Protection from degradation
 Increased translational
efficiency
 Transport to cytoplasm
 Splicing of first intron

85
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
86
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

87
 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 88
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 89
and termination
 What terminates
transcription by polymerase?

90
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
91
 RNA Pol I & III recognize
distinct promoters , using
The transcription in eukaryotes

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

92
Pol I promoter recognition

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

Fig 12-21 Pol I promoter region 93

Pol III promoter recognition
1. Different forms, 2. locates
downstream of the transcription site

TFIIIC binds to the promoter, recruiting

TFIIIB, which in turn recruits RNAP III

Fig 12-22 Pol III core promoter 94

Key points of the chapter
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), σ factor (4
domains), α CTD, 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 95
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) 96
(3)RNAP I and III transcription
---GTFs and promoter recognition,
formation of the initiation complex

97
 Homework

See the animations

Complete all the
excises on your
study CD. Enjoy it
98