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
2005-5-10
3
Part IV Regulation
Ch 16,Regulation in prokaryotes
Ch 17,Regulation in eukaryotes
Ch 18,Regulation during
development
Ch 19,Comparative genomics
and evolution of animal diversity
4
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.
Expression of many genes in
cells are regulated
5
Chapter 16 Regulation principles and
How genes are regulated in
bacteria
Chapter 17 Basic mechanism of gene
expression in eukaryotes
Chapter 18 How genes are regulated
to bestow cell type specificity in a
group of genetically identical cells
Chapter 19 How different animals
diverse in genomes,why human is
so special?
6
Surfing the contents of Part IV
7
Some of the peoples who
significantly contribute to the
knowledge of gene regulation
8
9
10
Chapter 16
Gene Regulation
in Prokaryotes
?Molecular Biology Course
11
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
12
Topic 1,Principles
of Transcription
Regulation
CHAPTER 16 Gene Regulation in Prokaryotes
5/10/2005
13
1-1 Gene Expression is Controlled
by Regulatory Proteins
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
Pri
nci
ple
s of
T
ran
scri
pti
on
Re
gul
ati
on
14
What are the
transcription steps
targeted by the
regulators?
15
Fig 12-3-initiation
Promoter Binding
(closed complex)
Promoter,melting”
(open complex)
Initial
transcription
16
Fig 12-3-Elongation and
termination
Termination
Elongation
17
1-2 Targeting promoter
binding,Many promoters are
regulated by activators that
help RNAP bind DNA and by
repressors that block the
binding
? At many promoters,RNAP binds weakly
? Lac operon is a good example
Pri
nci
ple
s of
T
ran
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on
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gul
ati
on
18
a,Absence of
Regulatory
Proteins
(operator)
b,To Control
Expression
c,To Activate
Expression
Fig 16-1
19
1-3 Targeting transition to the open
complex,Some Activators Work by
Allostery and Regulate Steps after
RNA Polymerase Binding
Fig 16-2
Examples,
Activator promoter
NtrC glnA
MerR merT
20
Some promoters are inefficient at
more than one step and can be
activated by more than one
mechanism
Repressors can work in ways other
than just blocking the promoter
binding,For example,inhibition of
the transition to the open complex.
21
1-6 Targeting termination and
beyond,Antitermination and
Beyond
?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.
Pri
nci
ple
s of
T
ran
scri
pti
on
Re
gul
ati
on
22
1-4 Action at a Distance and DNA
Looping,Some proteins interact with each
other even when bound to sites well separated
on the DNA
Fig 16-3
23
Fig 16-4 DNA-binding protein can
facilitate interaction between DNA-
binding proteins at a distance
Fig 16-4
24
1-5 Cooperative Binding and
Allostery have Many Roles in Gene
Regulation
?Cooperative binding,the activator
interacts simultaneously with DNA and
polymerase and so recruits the enzyme to
the promoter
?Group of regulators often bind DNA
cooperatively,(1) produce sensitive switches
to rapidly turn on a gene expression,(2)
integrate signals (some genes are activated
when multiple signals are present)
25
Allostery is not only a mechanism of
gene activation,it is also often the way
that regulators are controlled by their
specific signals.
26
Topic 2,Regulation
of Transcription
Initiation,
Examples
from Bacteria
CHAPTER 16 Gene Regulation in Prokaryotes
5/10/2005
27
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.
Sometimes are encoded by the gene under
the control of a different promoter
28
Control element
Structural genes
29
First example,Lac
operon
5/10/2005
Regulation of Transcription Initiation in Bacteria
30
The lactose (Lac) Operon
(乳糖操纵子 )
The enzymes required for the use of
lactose as a carbon source are only
synthesized when lactose is available
as the sole carbon source,
Fig 16-5
The LAC operon
31
Lactose operon,a regulatory gene and
3 stuctural genes,and 2 control elements
lacI
Regulatory gene
lacZ lacY lacA DNA
m-RNA
β -Galactosidase
Permease
Transacetylase
Protein
Structural GenesCis-acting
elements
PlacI P
lac Olac
The LAC operon
32
lacY encodes a cell membrane protein called lactose
permease (半乳糖苷渗透酶 ) to
transport Lactose across the
cell wall
lacZ codes for β-galactosidase (半乳糖苷酶 ) for lactose hydrolysis
lacA encodes a thiogalactoside
transacetylase (硫代半乳糖苷转
乙酰酶 )to get rid of the toxic
thiogalacosides
The LAC operon
33
1,The lacZ,lacY,lacA genes are
transcribed into a single lacZYA mRNA
(called polycistronic message) under the
control of a signal promoter Plac,
2,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
The LAC operon
34
2-1,An activator and a
repressor together control
the lac genes
The activator,CAP (Catabolite
Activator Protein) or CRP (cAMP
Receptor Protein); responses to the
glucose level.
The repressor,lac repressor that is
encoded by LacI gene; responses to
the lactose.
The LAC operon
35
Fig 16-6
The LAC operon
36
2-2,CAP and lac repressor
have opposing effects on
RNA polymerase binding to
the lac promoter
The LAC operon
37
The site bound by lac repressor is
called the lac operator,
The LAC operon
38
The lac operator overlaps promoter,
and so repressor bound to the
operator physically prevents RNA
polymerase from binding to the
promoter.
Fig 16-8
The LAC operon
39
?CAP binds to a site with the similar
structure as the operator,which is
60 bp upstream of the start site
of transcription.
?CAP also interacts with the enzyme
and recruit it to the promoter.
Fig 16-9
a CTD,C-terminal domain of the a subunit of RNAP
40
2-3,CAP has separate
activating and DNA-
binding surface
CAP binds
as a dimer a CTD
Fig 16-10
The LAC operon
41
2-4,CAP and lac repressor
bind DNA using a common
structural motif
The LAC operon
42
Both CAP and lac repressor bind DNA
using a helix-turn-helix motif.
One is the recognition helix that can fits
into the major groove of the DNA.
Fig 16-11
The LAC operon
43
DNA binding by a helix-turn-helix motif
Fig 16-12
Hydrogen
Bonds
between l
repressor
and the
major groove
of the
operator
The LAC operon
44
Lac repressor binds as a tetramer,with
each operator is contacted by a repressor
dimer,In addition to the primary
operator,there are two other lac
operators located 400 bp downstream and
90 bp upstream,respectively.
Not all the binding use a helix-turn-helix motif
Fig 16-13
45
2-5,The activity of Lac
repressor and CAP are
controlled allosterically by
their signals
Binding of the corresponding
signals alter the structure of
these two regulatory proteins
The LAC operon
46
i p o z y a
Very low level of lac mRNA
Absence of lactose
Active
i p o z y a
b-Galactosidase
Permease
Transacetylase
Presence of lactose
Inactive
Lack of inducer,the lac
repressor block all but a
very low level of trans-
cription of lacZYA,
Lactose is present,the low
basal level of permease
allows its uptake,andβ-
galactosidase catalyzes the
conversion of some lactose
to allolactose.
Allolactose acts as an
inducer,binding to the
lac repressor and inactivate
it,
Response to lactose
47
Response to glucose
The LAC operon
48
? 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.
2-6,Combinatorial Control
(组合调控 ),CAP controls other
genes as well
Regulation of Transcription Initiation in Bacteria
49
Second example,
Alternative s factor
5/10/2005
Regulation of Transcription Initiation in Bacteria
50
2-7,Alternative s factor
direct RNA polymerase to
alternative site of promoters
Alternative s factors
51
s factor:
s factor subunit bound to RNA
polymerase for transcription initiation
52
Promoter recognition
Different σfactors binding to the
same RNA Pol
Confer each of them a new promoter
specificity
s70 factors is most common one in E,
coli under the normal growth condition
Alternative s factors
53
Many bacteria produce alternative
sets of σfactors to meet the regulation
requirements of transcription under
normal and extreme growth condition
E,coli, Heat shock s32
Sporulation in Bacillus subtilis
Bacteriophage σfactors
Alternative s factors
54
Heat shock
? 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.
Alternative s factors
55
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,
Bacteriophages
Alternative s factors
56
B,subtilis SPO1 phage expresses
a cascade of σfactors which allow
a defined sequence of expression
of different phage genes,
Fig 16-14Alternative s factors
57
Normal bacterial holoenzyme
Express early genes
Encodeσfactor for transcription
of late genes
Encode σ28
Express middle genes (gene 34 and 33 )
Alternative s factors
58
Third example,NtrC
and MerR and
allosteric activation
5/10/2005
Regulation of Transcription Initiation in Bacteria
59
2-7,NtrC and MerR:
Transcriptional activators
that work by allostery rather
than by recruitment
NtrC and MerR and allosteric activation
60
?The majority of activators work by
recruitment,such as CAP,These
activators simply bring an active
form of RNA polymerase to the
promoter
?In this 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.
61
? In the absence of these activators,
the 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
62
2-8,NtrC has ATPase activity
and works from DNA sites
far from the gene
NtrC and MerR and allosteric activation
? NtrC controls expression of genes
involved in nitrogen metabolism,such
as the glnA gene
? NtrC has separate activating and
DNA-binding domains,and binds
DNA only when the nitrogen levels
are low.
63
Low nitrogen levels??NtrC phosphorylation
and conformational change??The DNA
binding domain binds DNA sites at ~ -150
position??NtrC interacts with s54 (glnA
promoter recognition)??ATP hydrolysis and
conformation change in polymerase??
transcription STARTs
Fig 16-15 activation by NtrC
64
2-8,MerR activates
transcription by twisting
promoter DNA
NtrC and MerR and allosteric activation
? 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,MerR
binds to a sequence between –10 and
–35 regions of the merT promoter
and activates merT expression.
65
As a s70 promoter,merT contains 19 bp
between –10 and –35 elements (the typical
length is 15-17 bp),leaving these two
elements neither optimally separated nor
aligned,
66Fig 16-15 Structure of a merT-like promoter
67
Fig 16-15
When Hg2+ is absent,MerR binds to the
promoter and locks it in the unfavorable
conformation
When Hg2+ is present,MerR binds Hg2+ and
undergo conformational change,which twists
the promoter to restore it to the structure
close to a strong s70 promoter
68
2-9,Some repressors hold
RNA polymerase at the
promoter rather than
excluding it
Regulation of Transcription Initiation in Bacteria
69
Repressors work in many ways,
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
Locking the promoter in a conformation
incompatible with transcription
initiation
70
Fourth example,
araBAD operon
5/10/2005
Regulation of Transcription Initiation in Bacteria
71
2-10,AraC and control of the
araBAD operon by antiactivation
The araBAD operon
? 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.
72
Different from the Lac operon,two
activators AraC and CAP work together
to activate the araBAD operon
expression
Fig 16-18
CAP site
194 bp
73
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?
74
Topic 3,Examples of
Gene Regulation
at Steps After
Transcription
Initiation
CHAPTER 16 Gene Regulation in Prokaryotes
5/10/2005
75
3-1,Amino acid biosynthetic
operons are controlled by
premature transcription
termination,the tryptophan
operon
Examples of Gene Regulation at Steps After Transcription Initiation
76
?The trp operon encodes five
structural genes required for
tryptophan 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
The TRP operon
77
The Trp repressor
（色氨酸阻遏物 ）
The TRP operon
78
1,Trp repressor is encoded by a separate
operon trpR,and specifically interacts
with the operator that overlaps with
the promoter sequence
2,The repressor can only bind to the
operator when it is complexed with
tryptophan,Therefore,Try is a co-
repressor and inhibits its own synthesis
through end-product inhibition (negative
feed-back regulation).
The TRP operon
Remember the lac repressor acts as
an inducer
79
The TRP operon3,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 )
80
trpR operon
trp operon
The TRP operon
81
Attenuation (衰减子 ), a
regulation at the transcription
termination step & a second
mechanism to confirm that
little tryptophan is available
? 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
The TRP operon
82
Fig 16-19
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,Transcription and translation in bacteria
are coupled,Therefore,synthesis of
the leader peptide immediately follows
the transcription of leader RNA.
2,The leader peptide contains two
tryptophan codons,If the tryptophan
level is very low,the ribosome will pause
at these sites.
3,Ribosome pause at these sites alter the
secondary structure of the leader RNA,
which eliminates the intrinsic terminator
structure and allow the successful
transcription of the trp operon,
84
Fig 16-20 The leader RNA and leader
peptide of the trp operon
85
Complementary 3:4
termination of
transcription
Complementary
2:3 Elongation
of transcription
Four regions (1,2,3,4) of the leader
sequence can base pair and form different
structures depending on the ribosome action
free leader
RNA
The TRP operon
86
Low Trp
High Trp
Fig 16-21
87
Importance of attenuation
1,A typical negative feed-back regulation
2,Use of both repression and attenuation
allows a fine tuning of the level of the
intracellular tryptophan.
3,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.
4,Provides an example of regulation
without the use of a regulatory protein,
but using RNA structure instead.
88
Box 4 Riboswitches
Riboswitches are regulatory RNA elements
that act as direct sensors of small
molecule metabolites to control gene
transcription or translation.
Some operate at the level of
transcription termination
Others operate at the level of
translation
Another kind responds to the uncharged
tRNA rather than responding to a
metabolite,Antitermination mechanism.
89
90
3-2,Ribosomal proteins are
translational repressors of
their own synthesis,a negative
feedback
Examples of Gene Regulation at Steps After Transcription Initiation
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
91
Strategies to meet the challenges:
1,Organization of the ribosomal proteins
to several operons,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 is at the level of
translation,not transcription.
92
Ribosomal protein operons
The protein that acts as
a translational repressor
of the other proteins is
shaded red.
Fig 16-22
93
Strategies to meet the challenges (cont):
4,For each operon,one (or two) ribosomal
proteins binds the mRNA near the
translation initiation sequence,preventing
the ribosome from binding and initiating
translation,
5,Repressing translation of the first gene
also prevents expression of some or all of
the rest,Are proteins processed after
translation?
6,The strategy is very sensitive,A few
unused molecule of protein L4,for example,
will shut down synthesis of that protein and
other proteins in this operon,
94
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 regulated mRNA
Fig 16-23
95
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
Key points of the chapter
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
2005-5-10
3
Part IV Regulation
Ch 16,Regulation in prokaryotes
Ch 17,Regulation in eukaryotes
Ch 18,Regulation during
development
Ch 19,Comparative genomics
and evolution of animal diversity
4
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.
Expression of many genes in
cells are regulated
5
Chapter 16 Regulation principles and
How genes are regulated in
bacteria
Chapter 17 Basic mechanism of gene
expression in eukaryotes
Chapter 18 How genes are regulated
to bestow cell type specificity in a
group of genetically identical cells
Chapter 19 How different animals
diverse in genomes,why human is
so special?
6
Surfing the contents of Part IV
7
Some of the peoples who
significantly contribute to the
knowledge of gene regulation
8
9
10
Chapter 16
Gene Regulation
in Prokaryotes
?Molecular Biology Course
11
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
12
Topic 1,Principles
of Transcription
Regulation
CHAPTER 16 Gene Regulation in Prokaryotes
5/10/2005
13
1-1 Gene Expression is Controlled
by Regulatory Proteins
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
Pri
nci
ple
s of
T
ran
scri
pti
on
Re
gul
ati
on
14
What are the
transcription steps
targeted by the
regulators?
15
Fig 12-3-initiation
Promoter Binding
(closed complex)
Promoter,melting”
(open complex)
Initial
transcription
16
Fig 12-3-Elongation and
termination
Termination
Elongation
17
1-2 Targeting promoter
binding,Many promoters are
regulated by activators that
help RNAP bind DNA and by
repressors that block the
binding
? At many promoters,RNAP binds weakly
? Lac operon is a good example
Pri
nci
ple
s of
T
ran
scri
pti
on
Re
gul
ati
on
18
a,Absence of
Regulatory
Proteins
(operator)
b,To Control
Expression
c,To Activate
Expression
Fig 16-1
19
1-3 Targeting transition to the open
complex,Some Activators Work by
Allostery and Regulate Steps after
RNA Polymerase Binding
Fig 16-2
Examples,
Activator promoter
NtrC glnA
MerR merT
20
Some promoters are inefficient at
more than one step and can be
activated by more than one
mechanism
Repressors can work in ways other
than just blocking the promoter
binding,For example,inhibition of
the transition to the open complex.
21
1-6 Targeting termination and
beyond,Antitermination and
Beyond
?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.
Pri
nci
ple
s of
T
ran
scri
pti
on
Re
gul
ati
on
22
1-4 Action at a Distance and DNA
Looping,Some proteins interact with each
other even when bound to sites well separated
on the DNA
Fig 16-3
23
Fig 16-4 DNA-binding protein can
facilitate interaction between DNA-
binding proteins at a distance
Fig 16-4
24
1-5 Cooperative Binding and
Allostery have Many Roles in Gene
Regulation
?Cooperative binding,the activator
interacts simultaneously with DNA and
polymerase and so recruits the enzyme to
the promoter
?Group of regulators often bind DNA
cooperatively,(1) produce sensitive switches
to rapidly turn on a gene expression,(2)
integrate signals (some genes are activated
when multiple signals are present)
25
Allostery is not only a mechanism of
gene activation,it is also often the way
that regulators are controlled by their
specific signals.
26
Topic 2,Regulation
of Transcription
Initiation,
Examples
from Bacteria
CHAPTER 16 Gene Regulation in Prokaryotes
5/10/2005
27
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.
Sometimes are encoded by the gene under
the control of a different promoter
28
Control element
Structural genes
29
First example,Lac
operon
5/10/2005
Regulation of Transcription Initiation in Bacteria
30
The lactose (Lac) Operon
(乳糖操纵子 )
The enzymes required for the use of
lactose as a carbon source are only
synthesized when lactose is available
as the sole carbon source,
Fig 16-5
The LAC operon
31
Lactose operon,a regulatory gene and
3 stuctural genes,and 2 control elements
lacI
Regulatory gene
lacZ lacY lacA DNA
m-RNA
β -Galactosidase
Permease
Transacetylase
Protein
Structural GenesCis-acting
elements
PlacI P
lac Olac
The LAC operon
32
lacY encodes a cell membrane protein called lactose
permease (半乳糖苷渗透酶 ) to
transport Lactose across the
cell wall
lacZ codes for β-galactosidase (半乳糖苷酶 ) for lactose hydrolysis
lacA encodes a thiogalactoside
transacetylase (硫代半乳糖苷转
乙酰酶 )to get rid of the toxic
thiogalacosides
The LAC operon
33
1,The lacZ,lacY,lacA genes are
transcribed into a single lacZYA mRNA
(called polycistronic message) under the
control of a signal promoter Plac,
2,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
The LAC operon
34
2-1,An activator and a
repressor together control
the lac genes
The activator,CAP (Catabolite
Activator Protein) or CRP (cAMP
Receptor Protein); responses to the
glucose level.
The repressor,lac repressor that is
encoded by LacI gene; responses to
the lactose.
The LAC operon
35
Fig 16-6
The LAC operon
36
2-2,CAP and lac repressor
have opposing effects on
RNA polymerase binding to
the lac promoter
The LAC operon
37
The site bound by lac repressor is
called the lac operator,
The LAC operon
38
The lac operator overlaps promoter,
and so repressor bound to the
operator physically prevents RNA
polymerase from binding to the
promoter.
Fig 16-8
The LAC operon
39
?CAP binds to a site with the similar
structure as the operator,which is
60 bp upstream of the start site
of transcription.
?CAP also interacts with the enzyme
and recruit it to the promoter.
Fig 16-9
a CTD,C-terminal domain of the a subunit of RNAP
40
2-3,CAP has separate
activating and DNA-
binding surface
CAP binds
as a dimer a CTD
Fig 16-10
The LAC operon
41
2-4,CAP and lac repressor
bind DNA using a common
structural motif
The LAC operon
42
Both CAP and lac repressor bind DNA
using a helix-turn-helix motif.
One is the recognition helix that can fits
into the major groove of the DNA.
Fig 16-11
The LAC operon
43
DNA binding by a helix-turn-helix motif
Fig 16-12
Hydrogen
Bonds
between l
repressor
and the
major groove
of the
operator
The LAC operon
44
Lac repressor binds as a tetramer,with
each operator is contacted by a repressor
dimer,In addition to the primary
operator,there are two other lac
operators located 400 bp downstream and
90 bp upstream,respectively.
Not all the binding use a helix-turn-helix motif
Fig 16-13
45
2-5,The activity of Lac
repressor and CAP are
controlled allosterically by
their signals
Binding of the corresponding
signals alter the structure of
these two regulatory proteins
The LAC operon
46
i p o z y a
Very low level of lac mRNA
Absence of lactose
Active
i p o z y a
b-Galactosidase
Permease
Transacetylase
Presence of lactose
Inactive
Lack of inducer,the lac
repressor block all but a
very low level of trans-
cription of lacZYA,
Lactose is present,the low
basal level of permease
allows its uptake,andβ-
galactosidase catalyzes the
conversion of some lactose
to allolactose.
Allolactose acts as an
inducer,binding to the
lac repressor and inactivate
it,
Response to lactose
47
Response to glucose
The LAC operon
48
? 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.
2-6,Combinatorial Control
(组合调控 ),CAP controls other
genes as well
Regulation of Transcription Initiation in Bacteria
49
Second example,
Alternative s factor
5/10/2005
Regulation of Transcription Initiation in Bacteria
50
2-7,Alternative s factor
direct RNA polymerase to
alternative site of promoters
Alternative s factors
51
s factor:
s factor subunit bound to RNA
polymerase for transcription initiation
52
Promoter recognition
Different σfactors binding to the
same RNA Pol
Confer each of them a new promoter
specificity
s70 factors is most common one in E,
coli under the normal growth condition
Alternative s factors
53
Many bacteria produce alternative
sets of σfactors to meet the regulation
requirements of transcription under
normal and extreme growth condition
E,coli, Heat shock s32
Sporulation in Bacillus subtilis
Bacteriophage σfactors
Alternative s factors
54
Heat shock
? 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.
Alternative s factors
55
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,
Bacteriophages
Alternative s factors
56
B,subtilis SPO1 phage expresses
a cascade of σfactors which allow
a defined sequence of expression
of different phage genes,
Fig 16-14Alternative s factors
57
Normal bacterial holoenzyme
Express early genes
Encodeσfactor for transcription
of late genes
Encode σ28
Express middle genes (gene 34 and 33 )
Alternative s factors
58
Third example,NtrC
and MerR and
allosteric activation
5/10/2005
Regulation of Transcription Initiation in Bacteria
59
2-7,NtrC and MerR:
Transcriptional activators
that work by allostery rather
than by recruitment
NtrC and MerR and allosteric activation
60
?The majority of activators work by
recruitment,such as CAP,These
activators simply bring an active
form of RNA polymerase to the
promoter
?In this 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.
61
? In the absence of these activators,
the 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
62
2-8,NtrC has ATPase activity
and works from DNA sites
far from the gene
NtrC and MerR and allosteric activation
? NtrC controls expression of genes
involved in nitrogen metabolism,such
as the glnA gene
? NtrC has separate activating and
DNA-binding domains,and binds
DNA only when the nitrogen levels
are low.
63
Low nitrogen levels??NtrC phosphorylation
and conformational change??The DNA
binding domain binds DNA sites at ~ -150
position??NtrC interacts with s54 (glnA
promoter recognition)??ATP hydrolysis and
conformation change in polymerase??
transcription STARTs
Fig 16-15 activation by NtrC
64
2-8,MerR activates
transcription by twisting
promoter DNA
NtrC and MerR and allosteric activation
? 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,MerR
binds to a sequence between –10 and
–35 regions of the merT promoter
and activates merT expression.
65
As a s70 promoter,merT contains 19 bp
between –10 and –35 elements (the typical
length is 15-17 bp),leaving these two
elements neither optimally separated nor
aligned,
66Fig 16-15 Structure of a merT-like promoter
67
Fig 16-15
When Hg2+ is absent,MerR binds to the
promoter and locks it in the unfavorable
conformation
When Hg2+ is present,MerR binds Hg2+ and
undergo conformational change,which twists
the promoter to restore it to the structure
close to a strong s70 promoter
68
2-9,Some repressors hold
RNA polymerase at the
promoter rather than
excluding it
Regulation of Transcription Initiation in Bacteria
69
Repressors work in many ways,
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
Locking the promoter in a conformation
incompatible with transcription
initiation
70
Fourth example,
araBAD operon
5/10/2005
Regulation of Transcription Initiation in Bacteria
71
2-10,AraC and control of the
araBAD operon by antiactivation
The araBAD operon
? 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.
72
Different from the Lac operon,two
activators AraC and CAP work together
to activate the araBAD operon
expression
Fig 16-18
CAP site
194 bp
73
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?
74
Topic 3,Examples of
Gene Regulation
at Steps After
Transcription
Initiation
CHAPTER 16 Gene Regulation in Prokaryotes
5/10/2005
75
3-1,Amino acid biosynthetic
operons are controlled by
premature transcription
termination,the tryptophan
operon
Examples of Gene Regulation at Steps After Transcription Initiation
76
?The trp operon encodes five
structural genes required for
tryptophan 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
The TRP operon
77
The Trp repressor
（色氨酸阻遏物 ）
The TRP operon
78
1,Trp repressor is encoded by a separate
operon trpR,and specifically interacts
with the operator that overlaps with
the promoter sequence
2,The repressor can only bind to the
operator when it is complexed with
tryptophan,Therefore,Try is a co-
repressor and inhibits its own synthesis
through end-product inhibition (negative
feed-back regulation).
The TRP operon
Remember the lac repressor acts as
an inducer
79
The TRP operon3,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 )
80
trpR operon
trp operon
The TRP operon
81
Attenuation (衰减子 ), a
regulation at the transcription
termination step & a second
mechanism to confirm that
little tryptophan is available
? 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
The TRP operon
82
Fig 16-19
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,Transcription and translation in bacteria
are coupled,Therefore,synthesis of
the leader peptide immediately follows
the transcription of leader RNA.
2,The leader peptide contains two
tryptophan codons,If the tryptophan
level is very low,the ribosome will pause
at these sites.
3,Ribosome pause at these sites alter the
secondary structure of the leader RNA,
which eliminates the intrinsic terminator
structure and allow the successful
transcription of the trp operon,
84
Fig 16-20 The leader RNA and leader
peptide of the trp operon
85
Complementary 3:4
termination of
transcription
Complementary
2:3 Elongation
of transcription
Four regions (1,2,3,4) of the leader
sequence can base pair and form different
structures depending on the ribosome action
free leader
RNA
The TRP operon
86
Low Trp
High Trp
Fig 16-21
87
Importance of attenuation
1,A typical negative feed-back regulation
2,Use of both repression and attenuation
allows a fine tuning of the level of the
intracellular tryptophan.
3,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.
4,Provides an example of regulation
without the use of a regulatory protein,
but using RNA structure instead.
88
Box 4 Riboswitches
Riboswitches are regulatory RNA elements
that act as direct sensors of small
molecule metabolites to control gene
transcription or translation.
Some operate at the level of
transcription termination
Others operate at the level of
translation
Another kind responds to the uncharged
tRNA rather than responding to a
metabolite,Antitermination mechanism.
89
90
3-2,Ribosomal proteins are
translational repressors of
their own synthesis,a negative
feedback
Examples of Gene Regulation at Steps After Transcription Initiation
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
91
Strategies to meet the challenges:
1,Organization of the ribosomal proteins
to several operons,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 is at the level of
translation,not transcription.
92
Ribosomal protein operons
The protein that acts as
a translational repressor
of the other proteins is
shaded red.
Fig 16-22
93
Strategies to meet the challenges (cont):
4,For each operon,one (or two) ribosomal
proteins binds the mRNA near the
translation initiation sequence,preventing
the ribosome from binding and initiating
translation,
5,Repressing translation of the first gene
also prevents expression of some or all of
the rest,Are proteins processed after
translation?
6,The strategy is very sensitive,A few
unused molecule of protein L4,for example,
will shut down synthesis of that protein and
other proteins in this operon,
94
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 regulated mRNA
Fig 16-23
95
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
Key points of the chapter
