Li Xiaoling
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Content
Chapter 1 Introduction
Chapter 2 The Structures of DNA and RNA
Chapter 3 DNA Replication
Chapter 4 DNA Mutation and Repair
Chapter 5 RNA Transcription
Chapter 6 RNA Splicing
Chapter 7 Translation
Chapter 8 The Genetic code
Chapter 9 Regulation in prokaryotes
Chapter 10 Regulation in Eukaryotes
2016/3/21
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EVALUATION (GRADING) SYSTEM
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 Final exam: 70 points
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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
EXPRESSION OF THE GENOME
Ch 5 : Transcription
Ch 6 : RNA Splicing
Ch 7 : Translation
Ch 8 : The Genetic code
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
7
REGULATION
Ch 9: Regulation in prokaryotes
Ch 10: Regulation in eukaryotes
8
Expression of many genes in
cells are regulated
Housekeeping genes: expressed
constitutively, essential for basic
processes involving in cell replication
and growth.
Inducible genes: expressed only when
they are activated by inducers or
cellular factors.
9
Chapter 9 Regulation principles and
How genes are regulated in bacteria
Chapter 10 Basic mechanism of gene
expression in eukaryotes
10
Surfing the contents of Part IV
--The heart of the frontier
biological disciplines
11
Some of the peoples who
significantly contribute to the
knowledge of gene regulation
12
13
14
•Molecular Biology Course
Chapter 9
Gene Regulation
in Prokaryotes
15
TOPIC 1 Principles of Transcriptional
Regulation [watch the animation]
TOPIC 2 Regulation of Transcription
Initiation: Examples from Bacteria
(Lac operon, alternative s factors, NtrC,MerR, Gal
rep, araBAD operon)
TOPIC 3 Examples of Gene
Regulation after Transcription
Initiation (Trp operon)
TOPIC 4 The Case of Phage λ:
Layers of Regulation
16
CHAPTER 9 Gene Regulation in Prokaryotes
Topic 1: Principles
of Transcription
Regulation
17
Principles of Transcription Regulation
1. GENE EXPRESSION IS
CONTROLLED BY REGULATORY
PROTEINS (调控蛋白)
Gene expression is very often controlled by
Extracellular Signals, which are communicated
to genes by regulatory proteins:
 Positive regulators or activators
INCREASE the transcription
 Negative regulators or repressors
DECREASE or ELIMINATE the transcription
18
Principles of Transcription Regulation
2. GENE EXPRESSION IS
CONTROLLED AT DIFFERENT
STAGES (基因表达可以发生在不同时期)
The bulk of gene regulation
takes place at the initiation of
transcription.
Some involve transcriptional
elongation/termination, RNA
processing, and translation of
the mRNA into protein.
19
FIG 9-3INITIATION
Promoter Binding
(closed complex)
Promoter “melting”
(open complex)
Promoter
escape/Initial
transcription
20
FIG 9-3-ELONGATION AND
TERMINATION
Elongation
Termination
21
Principles of Transcription Regulation
3. TARGETING PROMOTER BINDING:
MANY PROMOTERS ARE REGULATED BY ACTIVATORS
(激活蛋白) THAT HELP RNAP BIND DNA
(RECRUITMENT) AND BY REPRESSORS (阻遏蛋白)
THAT BLOCK THE BINDING.
RNAP binds many promoters weakly (?),
activators that contain two binding sites to bind
a DNA sequence and RNAP simultaneously can
enhance the RNAP affinity with the promoters,
and thus increases gene transcription.This is
called recruitment regulation (招募调控).
On the contrary, Repressors can bind to the
operator inside of the promoter region, which
prevents RNAP binding and the transcription 22
of
the target gene.
a. Absence of
Regulatory Proteins:
basal level
expression
Fig 9-1
b. Repressor
binding to the
operator represses
expression
c. Activator
binding activates
expression
23
Principles of Transcription Regulation
4 Targeting transition to the open complex: Allostery
regulation (异构调控) after the RNA Polymerase
Binding
In some cases, RNAP binds the
promoters efficiently, but no spontaneous
isomerization occurs to lead to the open
complex, resulting in no or low transcription.
Some activators can bind to the closed
complex, inducing conformational change in
either RNAP or DNA promoter, which
converts the closed complex to open complex
and thus promotes the transcription.
24
Allostery regulation
Fig 9-2
Allostery is not only a mechanism of gene activation ,
it is also often the way that regulators are controlled
by their specific signals.
25
Principles of Transcription Regulation
5 Targeting promoter escape by some repressors
Repressors can work in ways:
(1) blocking the promoter binding.
(2) blocking the transition to the open
complex.
(3) blocking promoter escape
26
Some promoters are inefficient at
more than one step and can be
activated by more than one
mechanism.
Activation mechanisms include
recruitment (招募) and allostery
(异构).
27
Principles of Transcription Regulation
6. Cooperative binding (recruitment)
and allostery have many roles in gene
regulation
For example: group of regulators often bind DNA
cooperatively (activators and/or repressors
interact with each other and with the DNA,
helping each other to bind near a gene they
regulated) :
(1) produce sensitive switches to rapidly turn on a
gene expression,
(2) integrate signals (some genes are activated when
multiple signals are present).
28
Principles of Transcription Regulation
7. Action at a Distance and DNA Looping. The
regulator proteins can function even binding at a
DNA site far away from the promoter region,
through protein-protein interaction and DNA looping.
Fig 9-3
29
Fig 9-4 DNA-binding protein can
facilitate interaction between DNAbinding proteins at a distance
Fig 9-4
30
CHAPTER 9 Gene Regulation in Prokaryotes
Topic 2: Regulation
of Transcription
Initiation :
Examples
from Bacteria
31
Operon: a unit of prokarytoic gene
expression and regulation which typically
includes:
1. Structural genes for enzymes in a
specific biosynthetic pathway whose
expression is coordinately controlled.
2. Control elements, such as operator
sequence.
3. Regulator gene(s) whose products
recognize the control elements.
32
Sometimes are encoded by the gene under
the control of a different promoter
Control element
Structural genes
33
Regulation of Transcription Initiation in Bacteria
First example: Lac
operon
The lactose Operon
34
1. Lactose operon contains a
regulatory gene and 3 structural genes,
and 2 control elements.
Fig 9-5
The enzymes encoded by lacZ, lacY, lacA
are required for the use of lactose as a
carbon source. These genes are only
transcribed at a high level when lactose is
35
available as the sole carbon source.
The LAC operon
lacZ codes for β-galactosidase (半乳
糖苷酶) for lactose hydrolysis
lacY encodes a cell membrane
protein called lactose
permease (半乳糖苷渗透酶) to
transport Lactose across the
cell wall
lacA encodes a thiogalactoside
transacetylase (硫代半乳糖苷转
乙酰酶)to get rid of the toxic
36
thiogalacosides
The LAC operon
THE
LACZ, LACY, LACA GENES
TRANSCRIBED
INTO
A
ARE
SINGLE LACZYA
MRNA (POLYCISTRONIC MRNA) UNDER
CONTROL
OF
A SIGNAL
PROMOTER
THE
PLAC .
LacZYA transcription unit contains an
operator site Olac
position between bases -5
and +21 at the 3’-end of Plac
Binds with the lac repressor
37
The LAC operon
2. An activator and a repressor
together control the Lac operon
expression
The activator: CAP (Catabolite
Activator Protein,代谢产物激活蛋白) or
CRP (cAMP Receptor Protein,cAMP受体蛋
白); responses to the glucose level.
The repressor: lac repressor that is
encoded by LacI gene; responses to
the lactose.
38
Sugar switch-off mechanism
The LAC operon
The LAC operon
Fig 9-6
39
The LAC operon
3. Lac repressor bound to the
operator prevents RNAP from
binding to the promoter
The site bound by lac repressor is
called the lac operator (Olac ), and the
Olac overlaps promoter (Plac). Therefore
repressor bound to the operator
physically prevents RNA polymerase
from binding to the promoter.
40
The LAC operon
The LAC operon
Fig 9-8
41
The LAC operon
4. CAP activates the Lac
transcription through recruitment of
RNAP to the weak Plac
CAP has two binding sites, one interacts
with the CAP site on the DNA near
promoter, and one interacts with RNAP.
This cooperative binding ensures that
RNAP effectively binds to Plac and
initiates transcription of LacZYA.
42
The LAC operon
CAP site has the similar structure as the operator,
which is 60 bp upstream of the start site of
transcription.
 CAP also interacts with the RNAP and recruit it to
the promoter.

Fig 9-9
a CTD: C-terminal domain of the a subunit of RNAP
43
The LAC operon
CAP binds
as a dimer
a CTD
Fig 9-10. CAP has separate
activating and DNA-binding surface
44
5. CAP and Lac repressor bind
DNA using a common structural
motif: helix-turn-helix motif
Fig 9-11
One is the recognition helix that can fits
into the major groove of the DNA.
The LAC operon
45
DNA binding by a helix-turn-helix motif
Fig 9-12
Hydrogen
Bonds between
l repressor and
the major
groove of the
operator.
46
Lac operon contains three operators: the primary
operator and two other operators located 400
bp downstream and 90 bp upstream.
Lac repressor binds as a tetramer (四聚体), with
each operator is contacted by a repressor dimer
(二聚体). respectively.
Fig 9-13
47
6 The activity of Lac repressor and
CAP are controlled allosterically by
their signals.
Allolactose: turn of Lac repressor
cAMP: turn on CAP
Lactose is converted to allolactose by bgalactosidase, therefore lactose can
indirectly turn off the repressor.
Glucose lowers the cellular cAMP level,
therefore, glucose indirectly turn off CAP.
48
The LAC operon
Response to lactose
Lack of inducer: the lac
repressor block all but a
very low level of transcription of lacZYA .
Absence of lactose
i
p
o
z
y
a
Active
Very low level of lac mRNA
When Lactose is present,
the low basal level of
permease allows its uptake,
and b-galactosidase
catalyzes the conversion of
some lactose to allolactose.
Allolactose acts as an
inducer, binding to the
lac repressor and inactivate
it.
Presence of lactose
i
p
o
z
y
a
Inactive
Permease
Transacetylase
b-Galactosidase
49
Response to glucose
50
7: Combinatorial Control (组合调
控): CAP controls other genes as
well
A
regulator (CAP) works together with
different repressor at different genes,
this is an example of Combinatorial
Control.
 In fact, CAP acts at more than 100
genes in E.coli, working with an array of
partners.
51
Regulation of Transcription Initiation in Bacteria
Second example:
Alternative s factor
Alternative s factor (可变s因子)
direct RNA polymerase to
alternative site of promoters
52
s factor subunit bound to RNA polymerase
for transcription initiation (Ch 12)
53
Different s factors binding to the
same RNAP, conferring each of
them a new promoter specificity.

s70 factors is most common one in
E. coli under the normal growth
condition

54
Many bacteria produce alternative
sets of σfactors to meet the regulation
requirements of transcription under
normal and extreme growth condition.
Bacteriophage has its own σfactors
E. coli : Heat shock s32
Bacteriophage σfactors
Sporulation in Bacillus subtilis
55
HEAT SHOCK (热休克)
Around 17 proteins are specifically expressed in E.
coli when the temperature is increased above 37ºC.
 These proteins are expressed through transcription
by RNA polymerase using an alternative s factor
s32 coded by rhoH gene. s32 has its own specific
promoter consensus sequences.

56
Alternative s factors
Bacteriophages
Many bacteriophages synthesize
their own σfactors to endow the
host RNA polymerase with a
different promoter specificity and
hence to selectively express their
own phage genes .
57
Alternative s factors
Alternative s factors
Fig 9-14
B. subtilis SPO1 phage expresses
a cascade of σfactors which allow
a defined sequence of expression
of different phage genes.
58
Regulation of Transcription Initiation in Bacteria
Third example: NtrC
and MerR and
allosteric activation
Transcriptional activators NtrC and MerR
work by allostery rather than by recruitment.
59
Review
 The majority of activators work by recruitment, such
as CAP. These activators simply bring an active form
of RNA polymerase to the promoter
 In the case of allosteric activation, RNAP initially
binds the promoter in an inactive complex, and the
activator triggers an allosteric change in that complex
to activate transcription.
60
In the absence of NtrC and MerR,
RNAP binds to the corresponding
promoter to form a closed stable
complex.
 NtrC activator induces a
conformational change in the enzyme,
triggering transition to the open
complex
 MerR activator causes the allosteric
effect on the DNA and triggers the
transition to the open complex
61

NtrC and MerR and allosteric activation
1. NtrC has ATPase activity and
works from DNA sites far from the
gene
NtrC controls expression of genes involved in
nitrogen metabolism (氮代谢), such as the glnA
gene
 NtrC has separate activating and DNA-binding
domains, and binds DNA only when the nitrogen
levels are low.

62
Low nitrogen levels (低水平氮)NtrC
phosphorylation and conformational change
NtrC (?) binds DNA sites at ~-150 positio as a
dimer NtrC (?) interacts with s54 (glnA
promoter recognition)  NtrC ATPase activity
provides energy needed to induce a conformation
change in polymerase transcription STARTs
Fig 9-15 activation by NtrC
63
NtrC and MerR and allosteric activation
2. MerR activates transcription by
twisting promoter DNA


MerR controls a gene called merT, which encodes
an enzyme that makes cells resistant to the toxic
effects of mercury (抗汞酶)
In the presence of mercury (汞), MerR binds to a
sequence between –10 and –35 regions of the merT
promoter and activates merT expression.
64
As a s70 promoter, merT contains 19 bp
between –10 and –35 elements (the typical
length is 15-17 bp), leaving these two
elements recognized by s70 neither
optimally separated nor aligned.
65
66
Fig 9-15 Structure of a merT-like promoter
When Hg2+ is absent, MerR binds to the
promoter and locks it in the unfavorable
conformation
When Hg2+ is present, MerR binds Hg2+ and
undergoes conformational change, which
twists the promoter to restore it to the
structure close to a strong s70 promoter
Fig 9-15
67
Repressors work in many ways-review
Blocking RNA polymerase binding through binding to a
site overlapping the promoter. Lac repressor
Blocking the transition from the closed to open
complex. Repressors bind to sites beside a promoter,
interact with polymerase bound at that promoter and
inhibit initiation. E.coli Gal repressor
Blocking the promoter escape. P4 protein interaction
with PA2c (bacteriophage f29 )
68
Regulation of Transcription Initiation in Bacteria
Fourth example:
araBAD operon
69
The araBAD operon
1. AraC and control of the araBAD
operon by anti-activation

The promoter of the araBAD operon from E. coli
is activated in the presence of arabinose (阿拉伯
糖) and the absence of glucose and directs
expression of genes encoding enzymes required
for arabinose metabolism. This is very similar to
the Lac operon.
70
Different from the Lac operon, two activators AraC and
CAP work together to activate the araBAD operon
expression
194 bp
CAP site
Fig 9-18
71
Because the magnitude of induction of the araBAD
promoter by arabinose is very large, the promoter
is often used in expression vector.
If fusing a gene to the araBAD promoter, the
expression of the gene can be easily controlled by
addition of arabinose（阿拉伯糖）.
What is an expression vector ? [The answer is in the
Methods part.]
72
CHAPTER 9 Gene Regulation in Prokaryotes
Topic 3: Examples of
Gene Regulation
at Steps After
Transcription
Initiation
73
Examples of Gene Regulation at Steps After Transcription Initiation
First example: the
tryptophan operon (色
胺酸操纵子)
74
1. Amino acid biosynthetic operons
are controlled by premature
transcription termination: the trp
operon
75
The TRP operon
The trp operon encodes five
structural genes required for
tryptophan (色胺酸) synthesis.

These genes are regulated to
efficiently express only when
tryptophan is limiting.

Two layers of regulation are involved:
(1) transcription repression by the
Trp repressor (initiation); (2)
attenuation

76
The TRP operon
The Trp repressor
（色氨酸阻遏物 ）
77
The TRP operon
1.
2.
Trp repressor is encoded by a separate
operon trpR, and specifically interacts
with the operator that overlaps with
the promoter sequence
The repressor can only bind to the
operator when it is complexed with
tryptophan. Therefore, Try is a corepressor and inhibits its own synthesis
through end-product inhibition (negative
feed-back regulation).
Remember the lac repressor acts as
an inducer
78
The
TRP operon
3. The repressor reduces transcription
initiation by around 70-fold, which is
much smaller than the binding of lac
repressor.
4.
The repressor is a dimer of two subunits
which has a structure with a central
core and two flexible DNA-reading
heads (carboxyl-terminal of each
subunit )
79
The TRP operon
trpR operon
trp operon
80
The TRP operon
Attenuation (衰减作用) : a regulation
at the transcription termination step
& a second mechanism to confirm
that little tryptophan is available
81


Repressor serves as the primary
switch to regulate the expression of
genes in the trp operon
Attenuation serves as the fine switch
to determine if the genes need to be
efficiently expressed
82
Fig 9-19
Transcription of the trp operon is
prematurally stopped if the tryptophan
level is not low enough, which results in
the production of a leader RNA of 161
nt. (WHY?)
83
1.
2.
3.
Transcription and translation in bacteria
are coupled (细菌体内的转录和翻译是偶联的).
Therefore, synthesis of the leader
peptide immediately follows the
transcription of leader RNA.
The leader peptide contains two
tryptophan codons. If the tryptophan
level is very low, the ribosome will pause
at these sites.
Ribosome pause at these sites alter the
secondary structure of the leader RNA,
which eliminates the intrinsic terminator
structure and allow the successful
84
transcription of the trp operon.
85
Fig 9-20 The leader RNA and leader peptide
High Trp
Complementary 3:4
termination of
transcription
Low Trp
Complementary
2:3 Elongation
of transcription
Fig 9-21
86
Importance of attenuation
1.
2.
3.
4.
A typical negative feed-back regulation
Use of both repression and attenuation
allows a fine tuning of the level of the
intracellular tryptophan.
Attenuation alone can provide robust
regulation: other amino acids operons
like his and leu have no repressors and
rely entirely on attenuation for their
regulation.
Provides an example of regulation
without the use of a regulatory protein,
87
but using RNA structure instead.
Examples of Gene Regulation at Steps After Transcription Initiation
Second example:
Riboswitches-a RNA
structure control
mechanism
Riboswitches are regulatory RNA
elements that act as direct sensors of
small molecule metabolites to control
gene transcription or translation.
88
Box 4
1.Riboswitches operating at the level of
transcription termination using an
Antitermination mechanism.
2.Riboswitches operating at the level of
translation, controlling the formation of
an RNA structure that masks the
ribosome binding site on mRNA.
89
代谢物
代谢物
90
Tucker1 and Breaker, Current Opinion in Structural Biology 2005, 15:342–3
The 2nd structures of 7 riboswitches
and metabolites that they sense
91
Examples of Gene Regulation at Steps After Transcription Initiation
Third example:
Ribosomal proteins
are translational
repressors of their
own synthesis: a
negative feedback
92
Challenges the ribosome protein synthesis
1. Each ribosome contains some 50
distinct proteins that must be made at
the same rate.
2. The rate of the ribosome protein
synthesis is tightly closed to the cell’s
growth rate.
93
Strategies to meet the challenges-Operon
1. Organization of the ribosomal proteins
to several operons (操纵子) , each
containing up to 11 ribosomal protein
genes
2. Some nonribosomal proteins whose
synthesis is also linked to growth rate
are contained in these operons,
including those for RNAP subunits a, b
and b’.
3. The primary control (主要调控) is at the
level of translation, not transcription.94
Ribosomal protein operons
The protein that acts as
a translational repressor
of the other proteins is
shaded red.
Fig 9-22
95
Strategies to meet the challenges (cont):
4.
5.
6.
For each operon, one (or two) ribosomal
proteins binds the mRNA near the
translation initiation sequence, preventing
the ribosome from binding and initiating
translation.
Repressing translation of the first gene
also prevents expression of some or all of
the rest.
The strategy is very sensitive. A few
unused molecule of protein L4, for example,
will shut down synthesis of that protein and
96
other proteins in this operon.
7. The mechanism of one ribosomal protein also
functions as a regulator of its own translation: the
protein binds to the similar sites on the ribosomal
RNA and to the regulatory RNA in its own mRNA.
Fig 9-23
97
Key points of the chapter
1. Principles of gene regulation. (1) The
targeted gene expression events; (2) the
mechanisms: by recruitment/exclusion or
allostery
2. Regulation of transcription initiation in
bacteria: the lac operon, alternative s
factors, NtrC, MerR, Gal rep, araBAD operon
3. Examples of gene regulation after
transcription initiation: the trp operon,
riboswitch, regulation of the synthesis of
ribosomal proteins
98