Control of Gene Expression
基因表达的调控
Necessity of gene expression
并非所有的基因都需要连续表达。不同的细胞，不同的细胞周期，同一个基因表达水平也不一样。
为适应不断改变的生活环境。
调控可以使生物在其所处的环境中以最优的方式生存。
Tow difference Genes
Housekeeping Gene(管家基因 ),是一些细胞维持正常细胞功能所需的基因，是连续表达的
Regulated gene(可调节基因 ),它们的活性是以细胞或生物体的需求来控制的。
Difference Levels is Gene
expression regulated
DNA rearrangements,programmed change of
DNA sequence position.
Transcriptional regulation,RNA synthesis
RNA processing,splicing
Stability of mRNA,half life
Translational control,peptide synthesis
Post translational control,enzymes
Regulation of protein activity vs,amount
Regulate protein activity
– Allostery变构效应
– Covalent modification
– Sequestration
Regulate the amount of protein
– Gene transcription
– RNA processing
– RNA turnover
– mRNA translation
– Protein processing,assembly,turnover
Difference between prokaryotic and
eukaryotic regulation
Transcription often is controlled at the stage of
initiation,It may also be controlled at termination to
prevent transcription from proceeding past a terminator
to the gene(s) beyond,This is the primary control
strategy for bacterial gene expression
In eukaryotic cells,processing of the RNA product
may be regulated at the stages of modification,splicing,
transport,or stability,In bacteria,an mRNA is in
principle available for translation as soon as it is
synthesized,and these stages of control are not
available
Regulation of Transcription in
Prokaryotes
原核生物转录的调控
Lactose Operon
Jacob and monod
乳糖操纵子
Key Terms
Cistron(顺反子 Gene 基因 ),是一段能编码多肽链的 DNA，包括前导肽 leader和尾随肽 trailer，以及分割编码区 exons的间隔序列。
Operon (操纵子，操纵元 ):是细菌 DNA中基因表达和调控的的单元，包括结构基因和控制单元。
Structural gene(结构基因 ),编码任何 RNA或 protein的 DNA序列，
product other than a regulator.
Repressor protein(阻遏蛋白 ),能够结合到 DNA or RNA上阻断
transcription or translation。
Operator(调控元件 ):是一段 DNA位点，它能结合 Repressor
protein 来阻断相邻 promoter 的起始转录。
Rgulator gene(调节基因 ):能编码结合到特定 DNA位点的蛋白质来调控 transcription。
Corepressor辅阻遏物,使阻遏蛋白具有活性或使活性蛋白失去活性的物质
Operons
An operon is a cluster of coordinately
regulated genes,It contains:
Structural genes,encode enzymes
Regulatory genes,encode repressors or
activators of expression
Regulatory sites,e.g,promoters,operators
the lac Operon
The biochemistry aspect
Bacteria can grow in a
minimum medium (N,C,
salts)
Different carbon sources
require a unique set of
metabolizing enzymes
two enzymes are required
for lactose metabolism,
-galactosidase
lactose permease
Galactose epimerase
Diauxic growth of E,coli on a mixture of lactose + glucose.
OOH
CH2OH
O
OH
CH2OH
O
HO
HO
OH
OH
galactose glucose
lactose
l a c t o s e
G l u c o s e G a l a c t o s e
e p i m e r a s e
g l y c o l y s i s
- g al ac t o s i d as e
If E.coli presented with glucose & lactose,use
mainly glucose until gone,then use lactose.
Lac Operon,Repression
Fig,7.3
Regulatory mechanisms
negative regulation负调控,repression阻遏
– negative induction regulation负控诱导
– negative repression regulation负控阻遏
positive regulation正调控,inductor诱导
– positive induction regulation正控诱导
– positive repression regulation正控阻遏
Positive vs negative control
Regulatory
protein is
present
Mutate
regulatory
gene to lose
function
Positive control
Negative control
Example of
regulatory
protein
Operon ON
Operon OFF
Operon OFF
Operon ON
Activator
Repressor
Catabolic vs,biosynthetic
operons
Absence of
Catabolic
enzymes
Biosynthetic
enzymes
Effect
Induced
Repressed
Operon
encodes Presence of Effect
Substrate
Product Product
Substrate Induced (derepressed)
Repressed
Inducible vs,repressible operons
Metabolite
Inducible
Repressible
Effect
ON
OFF
Type of
operon Presence of Operon
metabolite
Trp
lactose lac
trp
Defined by response of operon to a metabolite (small molecule).
metabolite
Examples
Negative control of the lac
operon
Induced (derepressed) lac
operon
lacZ lacY lacA
AUG UAA AUG UAA AUG UAA
Structural
genes &
regulatory
sites in
operon
Polycistronic
mRNA
-galactosidase lactose
permease
-galactoside
transacetylase
transcription
translation
Promoter
Operator
Repressed lac operon
lacZ lacY lacA
Promoter
OperatorlacI
lac repressor
Repressor binds to the operator in the absence of the inducer
(a metabolite of lactose),and blocks transcription of the lac
operon.
Induction of the lac operon by derepression
lacZ lacY lacA
Promoter
OperatorlacI
lacZ lacY lacA
Promoter
OperatorlacI
Operon is expressed
lac repressor no longer
Binds operator
Inducer (allolactose)
Inducers of the lac operon
Lactose,the substrate for the operon,is
converted to its isomer allolactose.
Allolactose is the natural inducer.
A gratuitous inducer induces the operon
but is not metabolized itself,
– e.g,isopropylthiogalactoside= IPTG
-10 +1 +10 +20
5’TGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACA
3’ACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGT
Interactions between operator and
repressor
Dyad axis
Constitutive mutations A TGTTA C T
T ACAAT G A
Nucleotides in contact with
repressor
Promoter
Positive control:,catabolite
repression”
Glucose is the preferred carbon source for E,coli.
Glucose causes repression of operons whose
products catalyze the metabolism of other carbon
sources,e.g,lac operon and lactose.
This is called catabolite repression.
In the absence of glucose,operons needed for
metabolism of other carbon sources are induced.
Catabolite repression is mediated by
cAMP and CAP
cAMP
– 3’,5’-cyclic adenosine monophosphate
– In presence of glucose,[cAMP] is about 10-7 M.
– In absence of glucose,[cAMP] increases to about 10-4
M.
Catabolite activator protein = CAP
– Is a dimer
– Binds cAMP
cAMP-CAP binds DNA adjacent to promoter and
stimulates transcription
CAP (catabolite activator protein) or
crp gene (cAMP receptor protein)
CAP only active when cAMP is bound
CAP-cAMP stimulates transcription by
promoting formation of closed complex:
RNAP + Promoter? RPc → RPo
RPc = Closed complex
RPo = Open complex
CAP-cAMP is a dimer that binds to a short
sequence (~20 bp) w/dyad symmetry
Binding site for cAMP-CAP
-70 -60 -50
5’ATGTGAGTTAGCTCACACATT
3’TACACTCAATCGAGTGTGTAA
Dyad axis
Mutations that make
promoter nonresponsive
to CAP
A T
T A
Nucleotides in contact
with cAMP-CAP
Promoter
lac regulatory region
Repressor
TATAATUV5 mutation,up
Promoter OperatorActivator binding site
+11+1-10-35-52-72
TTTACA TATGTT
RNA polymerase
'a
scAMP-CAP
Some generalities
Repressors,activators and polymerases interact
primarily with one face of the DNA double helix.
Regulatory protein are frequently symmetrical and
bind to symmetrical sites on the DNA.
RNA polymerases are not symmetrical,and bind
to asymmetric sites,This helps establish the
direction of transcription.
cAMP-CAP helps RNA
polymerase bind to promoter by
interacting with the alpha subunit
More in chapter II of Part Four
CAP bound to DNA
The trp operon
The trp operon is responsible for the biosynthesis of
tryptophan.
In man and plants the amino acid is essential as we are
unable to synthesize this amino acid,so we obtain it
from food and our gut flora.
The trp operon is involved in biosyntheis,so it is
repressible by the presence of tryptophan
(N.B,catabolic steps are inducible,and anabolic steps
are repressible,by the products the operons are named
after).
trp operon
In the presence of excess tryptophan the trp operon is
turned off or REPRESSED.
Should the [tryptophan] decease then the trp operon is
expressed,
Tryptophan itself acts as a CO-REPRESSOR,and
unlike the lac operon does not induce expression.
Moreover there is no need for the additional CAP-
cAMP regulatory system,
trp operon
Tryptophan is synthesized in five steps from chorismate,
and each step is catalysed by one of five enzymes encoded
by trpA,B,C,D & E which are expressed as a polycistronic
mRNA,These five genes constitute the structural genes,
In addition there is a regulatory region the,
-OPERATOR,
and a regulatory protein,
- TRP REPRESSOR or APOREPRESSOR
(the aporepressor denotes that it cannot bind the attenuator
on its own in the absence of trytophan,but will in the
presence of tryptophan)
Similar to on-off of lac operon but with subtle differences!
The trp repressor/aporepressor
The trp repressor is encoded by the trpR gene.
Unlike the lac operon the trpR gene is not physically
close to the structural genes of the trp operon.
Aporepressor No repression
Transcription occurs
Aporepressor Active repressor
+ tryptophan NO transcription
Tryptophan increases the binding affinity of the
aporepressor for the operator
The trp operon
P = promoter
T = terminator
O = operator
trpR trpAP OP T T
Polycistronic mRNA (encodes 5 proteins)mRNA
TrpR (repressor)
5 separate proteins that were
synthesized from one mRNA
trpBtrpCtrpDtrpE
Attenuator
TrpR
The trp operon
Unlike the lac operon a simple on-off regulation of expression is
not optimal for an essential biosynthetic pathway,The need for
tryptophan may differ under different growth conditions,what is
fine for cells in lag phase may not be adequate for cells in log
growth.
Expression is therefore linked to the [tryptophan amino amino
acid] by a fine control mechanism called ATTENUATION
This fine control over-rides the transcriptional control by the
aporepressor,In effect,transcription is prematurely terminated if
[tryptophan] is potentially limiting.
Attenuation is a feature of most biosynthetic operons
Attenuation in the trp operon
Effectively adds a fine tuning to the regulation of the
trp operon.
Several key points:
1,Transcription & translation are tightly coupled in
bacteria (attenuation requires this).
2,Synthesis of a leader sequence rich in Trp (2 out of
14 aa) influences whether transcription of the trp
operon is complete.
3,If [Trp] is adequate transcription is terminated before
the trp operon.
4,If [Trp] is inadequate transcription is completed.
5,Termination of transcription is determined by leader
mRNA sequence.
Attenuation – a transcriptional form of control
mRNA leader sequence
Attenuator
110 140 trpE
Leader polypeptide
14 aa with 2 Trp aa
1 162
Typical stem loop
of Termination site
Genetic analysis of the trp operon
As with the lac operon mutation of the regulatory( trpR)
gene leads to enhanced expression of the trp operon.
Deletion of the attenuator region also leads to six-fold
enhanced expression of the trp operon in the presence or
absence of tryptophan.
The attenuator is not where the TrpR repressor binds.
This confirms the attenuator has a regulatory role.
Attenuation
1 2 3 4
42 31
mRNA
Trp codons
mRNA sections
1 base pairs with 2
3 base pairs with 4
ONLY 3 + 4 generate
the termination site
Attenuation – Inadequate [Trp]
1 2 3 4
mRNA
Trp codons
2 3
1 4
Ribosome stalls
due to low [Trp]
This large stem loop of 2 +
3 does NOT act as a
terminator,
Transcription continues!!
RNA polymerase
Attenuation – Adequate [Trp]
1 2 3 4
43
1
Ribosome moves
Rapidly along mRNA
mRNA sections
3 base pairs with 4 to
form a termination site,
such that RNApolymerase
prematurely falls off the
mRNA and aborts further
transcription,
mRNA
Summary of attenuation
The presence or absence of the TrpR repressor determines
whether or not the trp operon is transcribed.
However,when the trp operon is being transcribed the
[Trp] determines the speed at which the ribosome moves,
This in turn influences formation of the termination site by
sections 3 + 4,which influences whether or not
transcription is prematurely terminated!
Think of attenuation as a dimmer switch,gives fine
control,when the light (=gene expression) is on.
Attenuation vs Repression
For the trp operon repression is thought to be
responsible for 80-fold regulation.
Attenuation is thought to give a further 6-8 fold
regulation.
The combined regulation is nearly 500-fold for this
operon.
In other biosynthetic operons attenuation is the only
form of regulation as they lack a repressor-operator
organisation,
A comparison of amino acid leader sequences
Operon Sequence
trp Met Lys Arg Ile Phe Val Leu Lys Gly Trp Trp Arg Thr Ser
his Met Thr - - - - (His)7-----
Phe Met Lys - - - - (Phe)3-- (Phe)3--
Attenuation is a common feature of biosynthetic operons.
The amino acid being synthesized frequently features in
the leader sequence,which empasizes how important the
transcription-translation couple is!
Comparison of lac & trp operons
lac operon
encodes catabolic
enzymes.
lactose (allolactose) is an
inducer.
CAP regulation is
involved
lacI gene situated just
upstream of lacZYA gene
cluster
No transcriptional
termination
trp operon
encodes anabolic
enzymes.
tryptophan is an co-
repressor.
No CAP involvement
trpR gene situated a long
way away from trp gene
cluster
See transcriptional
termination as a fine
control
Differences between the trp and lac operon
The differences are as follows:
1,Tryptophan acts directly in the repression system rather
than as an inducer,This is a key difference between
anabolic (biosynthesis) and catabolic (degradative)
operons.
2,A simple on-off system for regulation of biosynthesis is
not adequate to give fine control for biosynthesis.
负调控 正调控
L ac O A ra O
诱导 失活的阻遏物 活化的激活蛋白阻遏物 诱导物 失活的活性蛋白 诱导物阻遏 诱导 阻遏 诱导
T r p O
阻遏失活的活性蛋白辅 - 阻遏物 活化的激活蛋白 辅 - 阻遏物 失活的活性蛋白诱导 阻遏 诱导 阻遏图 1 6 - 1 原核生物结构基因的 4 种表达调控类型 ( 仿 B,L ew i n,,G E N E S,Ⅵ,1 9 9 7,Fi g,1 2,2 1 )
The End
基因表达的调控
Necessity of gene expression
并非所有的基因都需要连续表达。不同的细胞，不同的细胞周期，同一个基因表达水平也不一样。
为适应不断改变的生活环境。
调控可以使生物在其所处的环境中以最优的方式生存。
Tow difference Genes
Housekeeping Gene(管家基因 ),是一些细胞维持正常细胞功能所需的基因，是连续表达的
Regulated gene(可调节基因 ),它们的活性是以细胞或生物体的需求来控制的。
Difference Levels is Gene
expression regulated
DNA rearrangements,programmed change of
DNA sequence position.
Transcriptional regulation,RNA synthesis
RNA processing,splicing
Stability of mRNA,half life
Translational control,peptide synthesis
Post translational control,enzymes
Regulation of protein activity vs,amount
Regulate protein activity
– Allostery变构效应
– Covalent modification
– Sequestration
Regulate the amount of protein
– Gene transcription
– RNA processing
– RNA turnover
– mRNA translation
– Protein processing,assembly,turnover
Difference between prokaryotic and
eukaryotic regulation
Transcription often is controlled at the stage of
initiation,It may also be controlled at termination to
prevent transcription from proceeding past a terminator
to the gene(s) beyond,This is the primary control
strategy for bacterial gene expression
In eukaryotic cells,processing of the RNA product
may be regulated at the stages of modification,splicing,
transport,or stability,In bacteria,an mRNA is in
principle available for translation as soon as it is
synthesized,and these stages of control are not
available
Regulation of Transcription in
Prokaryotes
原核生物转录的调控
Lactose Operon
Jacob and monod
乳糖操纵子
Key Terms
Cistron(顺反子 Gene 基因 ),是一段能编码多肽链的 DNA，包括前导肽 leader和尾随肽 trailer，以及分割编码区 exons的间隔序列。
Operon (操纵子，操纵元 ):是细菌 DNA中基因表达和调控的的单元，包括结构基因和控制单元。
Structural gene(结构基因 ),编码任何 RNA或 protein的 DNA序列，
product other than a regulator.
Repressor protein(阻遏蛋白 ),能够结合到 DNA or RNA上阻断
transcription or translation。
Operator(调控元件 ):是一段 DNA位点，它能结合 Repressor
protein 来阻断相邻 promoter 的起始转录。
Rgulator gene(调节基因 ):能编码结合到特定 DNA位点的蛋白质来调控 transcription。
Corepressor辅阻遏物,使阻遏蛋白具有活性或使活性蛋白失去活性的物质
Operons
An operon is a cluster of coordinately
regulated genes,It contains:
Structural genes,encode enzymes
Regulatory genes,encode repressors or
activators of expression
Regulatory sites,e.g,promoters,operators
the lac Operon
The biochemistry aspect
Bacteria can grow in a
minimum medium (N,C,
salts)
Different carbon sources
require a unique set of
metabolizing enzymes
two enzymes are required
for lactose metabolism,
-galactosidase
lactose permease
Galactose epimerase
Diauxic growth of E,coli on a mixture of lactose + glucose.
OOH
CH2OH
O
OH
CH2OH
O
HO
HO
OH
OH
galactose glucose
lactose
l a c t o s e
G l u c o s e G a l a c t o s e
e p i m e r a s e
g l y c o l y s i s
- g al ac t o s i d as e
If E.coli presented with glucose & lactose,use
mainly glucose until gone,then use lactose.
Lac Operon,Repression
Fig,7.3
Regulatory mechanisms
negative regulation负调控,repression阻遏
– negative induction regulation负控诱导
– negative repression regulation负控阻遏
positive regulation正调控,inductor诱导
– positive induction regulation正控诱导
– positive repression regulation正控阻遏
Positive vs negative control
Regulatory
protein is
present
Mutate
regulatory
gene to lose
function
Positive control
Negative control
Example of
regulatory
protein
Operon ON
Operon OFF
Operon OFF
Operon ON
Activator
Repressor
Catabolic vs,biosynthetic
operons
Absence of
Catabolic
enzymes
Biosynthetic
enzymes
Effect
Induced
Repressed
Operon
encodes Presence of Effect
Substrate
Product Product
Substrate Induced (derepressed)
Repressed
Inducible vs,repressible operons
Metabolite
Inducible
Repressible
Effect
ON
OFF
Type of
operon Presence of Operon
metabolite
Trp
lactose lac
trp
Defined by response of operon to a metabolite (small molecule).
metabolite
Examples
Negative control of the lac
operon
Induced (derepressed) lac
operon
lacZ lacY lacA
AUG UAA AUG UAA AUG UAA
Structural
genes &
regulatory
sites in
operon
Polycistronic
mRNA
-galactosidase lactose
permease
-galactoside
transacetylase
transcription
translation
Promoter
Operator
Repressed lac operon
lacZ lacY lacA
Promoter
OperatorlacI
lac repressor
Repressor binds to the operator in the absence of the inducer
(a metabolite of lactose),and blocks transcription of the lac
operon.
Induction of the lac operon by derepression
lacZ lacY lacA
Promoter
OperatorlacI
lacZ lacY lacA
Promoter
OperatorlacI
Operon is expressed
lac repressor no longer
Binds operator
Inducer (allolactose)
Inducers of the lac operon
Lactose,the substrate for the operon,is
converted to its isomer allolactose.
Allolactose is the natural inducer.
A gratuitous inducer induces the operon
but is not metabolized itself,
– e.g,isopropylthiogalactoside= IPTG
-10 +1 +10 +20
5’TGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACA
3’ACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGT
Interactions between operator and
repressor
Dyad axis
Constitutive mutations A TGTTA C T
T ACAAT G A
Nucleotides in contact with
repressor
Promoter
Positive control:,catabolite
repression”
Glucose is the preferred carbon source for E,coli.
Glucose causes repression of operons whose
products catalyze the metabolism of other carbon
sources,e.g,lac operon and lactose.
This is called catabolite repression.
In the absence of glucose,operons needed for
metabolism of other carbon sources are induced.
Catabolite repression is mediated by
cAMP and CAP
cAMP
– 3’,5’-cyclic adenosine monophosphate
– In presence of glucose,[cAMP] is about 10-7 M.
– In absence of glucose,[cAMP] increases to about 10-4
M.
Catabolite activator protein = CAP
– Is a dimer
– Binds cAMP
cAMP-CAP binds DNA adjacent to promoter and
stimulates transcription
CAP (catabolite activator protein) or
crp gene (cAMP receptor protein)
CAP only active when cAMP is bound
CAP-cAMP stimulates transcription by
promoting formation of closed complex:
RNAP + Promoter? RPc → RPo
RPc = Closed complex
RPo = Open complex
CAP-cAMP is a dimer that binds to a short
sequence (~20 bp) w/dyad symmetry
Binding site for cAMP-CAP
-70 -60 -50
5’ATGTGAGTTAGCTCACACATT
3’TACACTCAATCGAGTGTGTAA
Dyad axis
Mutations that make
promoter nonresponsive
to CAP
A T
T A
Nucleotides in contact
with cAMP-CAP
Promoter
lac regulatory region
Repressor
TATAATUV5 mutation,up
Promoter OperatorActivator binding site
+11+1-10-35-52-72
TTTACA TATGTT
RNA polymerase
'a
scAMP-CAP
Some generalities
Repressors,activators and polymerases interact
primarily with one face of the DNA double helix.
Regulatory protein are frequently symmetrical and
bind to symmetrical sites on the DNA.
RNA polymerases are not symmetrical,and bind
to asymmetric sites,This helps establish the
direction of transcription.
cAMP-CAP helps RNA
polymerase bind to promoter by
interacting with the alpha subunit
More in chapter II of Part Four
CAP bound to DNA
The trp operon
The trp operon is responsible for the biosynthesis of
tryptophan.
In man and plants the amino acid is essential as we are
unable to synthesize this amino acid,so we obtain it
from food and our gut flora.
The trp operon is involved in biosyntheis,so it is
repressible by the presence of tryptophan
(N.B,catabolic steps are inducible,and anabolic steps
are repressible,by the products the operons are named
after).
trp operon
In the presence of excess tryptophan the trp operon is
turned off or REPRESSED.
Should the [tryptophan] decease then the trp operon is
expressed,
Tryptophan itself acts as a CO-REPRESSOR,and
unlike the lac operon does not induce expression.
Moreover there is no need for the additional CAP-
cAMP regulatory system,
trp operon
Tryptophan is synthesized in five steps from chorismate,
and each step is catalysed by one of five enzymes encoded
by trpA,B,C,D & E which are expressed as a polycistronic
mRNA,These five genes constitute the structural genes,
In addition there is a regulatory region the,
-OPERATOR,
and a regulatory protein,
- TRP REPRESSOR or APOREPRESSOR
(the aporepressor denotes that it cannot bind the attenuator
on its own in the absence of trytophan,but will in the
presence of tryptophan)
Similar to on-off of lac operon but with subtle differences!
The trp repressor/aporepressor
The trp repressor is encoded by the trpR gene.
Unlike the lac operon the trpR gene is not physically
close to the structural genes of the trp operon.
Aporepressor No repression
Transcription occurs
Aporepressor Active repressor
+ tryptophan NO transcription
Tryptophan increases the binding affinity of the
aporepressor for the operator
The trp operon
P = promoter
T = terminator
O = operator
trpR trpAP OP T T
Polycistronic mRNA (encodes 5 proteins)mRNA
TrpR (repressor)
5 separate proteins that were
synthesized from one mRNA
trpBtrpCtrpDtrpE
Attenuator
TrpR
The trp operon
Unlike the lac operon a simple on-off regulation of expression is
not optimal for an essential biosynthetic pathway,The need for
tryptophan may differ under different growth conditions,what is
fine for cells in lag phase may not be adequate for cells in log
growth.
Expression is therefore linked to the [tryptophan amino amino
acid] by a fine control mechanism called ATTENUATION
This fine control over-rides the transcriptional control by the
aporepressor,In effect,transcription is prematurely terminated if
[tryptophan] is potentially limiting.
Attenuation is a feature of most biosynthetic operons
Attenuation in the trp operon
Effectively adds a fine tuning to the regulation of the
trp operon.
Several key points:
1,Transcription & translation are tightly coupled in
bacteria (attenuation requires this).
2,Synthesis of a leader sequence rich in Trp (2 out of
14 aa) influences whether transcription of the trp
operon is complete.
3,If [Trp] is adequate transcription is terminated before
the trp operon.
4,If [Trp] is inadequate transcription is completed.
5,Termination of transcription is determined by leader
mRNA sequence.
Attenuation – a transcriptional form of control
mRNA leader sequence
Attenuator
110 140 trpE
Leader polypeptide
14 aa with 2 Trp aa
1 162
Typical stem loop
of Termination site
Genetic analysis of the trp operon
As with the lac operon mutation of the regulatory( trpR)
gene leads to enhanced expression of the trp operon.
Deletion of the attenuator region also leads to six-fold
enhanced expression of the trp operon in the presence or
absence of tryptophan.
The attenuator is not where the TrpR repressor binds.
This confirms the attenuator has a regulatory role.
Attenuation
1 2 3 4
42 31
mRNA
Trp codons
mRNA sections
1 base pairs with 2
3 base pairs with 4
ONLY 3 + 4 generate
the termination site
Attenuation – Inadequate [Trp]
1 2 3 4
mRNA
Trp codons
2 3
1 4
Ribosome stalls
due to low [Trp]
This large stem loop of 2 +
3 does NOT act as a
terminator,
Transcription continues!!
RNA polymerase
Attenuation – Adequate [Trp]
1 2 3 4
43
1
Ribosome moves
Rapidly along mRNA
mRNA sections
3 base pairs with 4 to
form a termination site,
such that RNApolymerase
prematurely falls off the
mRNA and aborts further
transcription,
mRNA
Summary of attenuation
The presence or absence of the TrpR repressor determines
whether or not the trp operon is transcribed.
However,when the trp operon is being transcribed the
[Trp] determines the speed at which the ribosome moves,
This in turn influences formation of the termination site by
sections 3 + 4,which influences whether or not
transcription is prematurely terminated!
Think of attenuation as a dimmer switch,gives fine
control,when the light (=gene expression) is on.
Attenuation vs Repression
For the trp operon repression is thought to be
responsible for 80-fold regulation.
Attenuation is thought to give a further 6-8 fold
regulation.
The combined regulation is nearly 500-fold for this
operon.
In other biosynthetic operons attenuation is the only
form of regulation as they lack a repressor-operator
organisation,
A comparison of amino acid leader sequences
Operon Sequence
trp Met Lys Arg Ile Phe Val Leu Lys Gly Trp Trp Arg Thr Ser
his Met Thr - - - - (His)7-----
Phe Met Lys - - - - (Phe)3-- (Phe)3--
Attenuation is a common feature of biosynthetic operons.
The amino acid being synthesized frequently features in
the leader sequence,which empasizes how important the
transcription-translation couple is!
Comparison of lac & trp operons
lac operon
encodes catabolic
enzymes.
lactose (allolactose) is an
inducer.
CAP regulation is
involved
lacI gene situated just
upstream of lacZYA gene
cluster
No transcriptional
termination
trp operon
encodes anabolic
enzymes.
tryptophan is an co-
repressor.
No CAP involvement
trpR gene situated a long
way away from trp gene
cluster
See transcriptional
termination as a fine
control
Differences between the trp and lac operon
The differences are as follows:
1,Tryptophan acts directly in the repression system rather
than as an inducer,This is a key difference between
anabolic (biosynthesis) and catabolic (degradative)
operons.
2,A simple on-off system for regulation of biosynthesis is
not adequate to give fine control for biosynthesis.
负调控 正调控
L ac O A ra O
诱导 失活的阻遏物 活化的激活蛋白阻遏物 诱导物 失活的活性蛋白 诱导物阻遏 诱导 阻遏 诱导
T r p O
阻遏失活的活性蛋白辅 - 阻遏物 活化的激活蛋白 辅 - 阻遏物 失活的活性蛋白诱导 阻遏 诱导 阻遏图 1 6 - 1 原核生物结构基因的 4 种表达调控类型 ( 仿 B,L ew i n,,G E N E S,Ⅵ,1 9 9 7,Fi g,1 2,2 1 )
The End
