分子生物學 第六章

分子生物學第六章第6章

基因表達調(diào)控(Controlling

of

the

Gene

Expression)(Source:Pique,Michaeland

Peter

E.Wright,Dept.(cover

photo,science

245

(11Aug

1989).))6.1.

transcriptionallevel

control6.2.

post-transcriptional

level

control6.4.

染色質(zhì)重建對基因表達的控制

expression

control

in

translationlevel

Death

(PCD)

anddevelopment闡明的科學問題真核與原核生物如何調(diào)控數(shù)以千、萬計的基因以最為經(jīng)濟、有效的時空模式進行轉(zhuǎn)錄，從而實現(xiàn)對環(huán)境的適應、細胞的分化,

組織的特化

和個體的發(fā)育。基因表達調(diào)控的簡介調(diào)控機理上調(diào)控層次上原核生物與真核生物基因表達調(diào)控機制具有驚人的相似性

共同的起源與共同的分子基礎

核酸分子間的互作核酸與蛋白質(zhì)分子間的互作蛋白質(zhì)分子間的互作

transcriptionallevel

post—transcriptionallevel

translationallevel

post—translationallevel基因表達的調(diào)控涉及RNA轉(zhuǎn)錄的開/關數(shù)量選擇性加工蛋白質(zhì)翻譯速率數(shù)量加工、降解和分泌…

轉(zhuǎn)錄水平上的調(diào)控是最為經(jīng)濟,

靈活，又是最為重要，復雜的調(diào)控●在復雜的基因組內(nèi)，確定需要基因轉(zhuǎn)錄的起始位點●精細調(diào)節(jié)基因表達的水平,以保證生物體對環(huán)境的適應●cis

factor&

trans

factor間嚴格而又靈活的互作●保證RNApolymerase

的進行式轉(zhuǎn)錄（不中斷，準確終止）遺傳信息的概念

C

>

c

10%;

結(jié)構(gòu)基因的編碼序列

triplet

codon（I類遺傳信息）90%;

重復，調(diào)節(jié)序列,Non

coding

RNA…

基因選擇性表達指令

重要的遺傳信息（II類遺傳信息）全基因組遺傳信息表達的方式?

組成型表達（constitutiveexpression）Housekeeping

gene?

誘導型表達（inducible

expression

by

signaling

molecular）Luxury

gene?

順、反因子間和反式因之間的互作方式Epistasis

effect6.1.

transcriptional

level

control

Prok.

operon

stringent

response

attenuator

transposon…6.1.1.Operon

control

(1961

Jacob.&

Monod.)a)

operon

concept●

控制某一代謝途徑的相關基因,緊密連鎖地排列在一起,受同一操縱子控制●●

●各結(jié)構(gòu)基因按一定比例協(xié)調(diào)翻譯

(

Z

:

Y

:A=

5

:

2

:

1

)具有極性突變效應

P&

O基因(cis)緊密連鎖或

彼此重疊

I

基因(trans)

位點不固定Lac

operonI

p

o

Z

Y

Ab)

operon

control

type

Negative

&

Positive?

I

geneNeg.Pos.i-

or

不加入I基因產(chǎn)物I+

or

加入I基因產(chǎn)物i-

or

不加入I基因產(chǎn)物I+

or

加入I基因產(chǎn)物operonon

operonoff

operonoff

operon

onNegativePositiverepressor

expressor

(apoinducer)

無輔基誘導物●Repressorbinding

on

O

site

阻止轉(zhuǎn)錄啟動

(來源：分子生物學（2007），鄭用璉，第239頁)●Operon

off●Negative

control

是廣泛保險的機制

（自然選擇使Prok.

獲得選擇優(yōu)勢）Positive

control

是靈活，嚴格，經(jīng)濟的調(diào)控機制意味轉(zhuǎn)錄效率極低?

Expressorbinding

front

p

site

激活轉(zhuǎn)錄啟動c)

Model●Signal

molecular

be

needed

forboth

typesAdd

signal

mol.

Operon

on

(inducible

operon)

InducerAdd

signal

mol.

Operon

off

(repressibleoperon)

Co-repressorOperon

control

modelnegativepositiveInd.

Rep.

inactiveactive

activeinactive(來源：分子生物學（2007），鄭用璉，第218頁)例

(分解酶類lactoseoperon)

I

gene

active

repressor

38

kd

/

monomer

tetramer

152

kd

binding

on

OperatorI+mut.

iC

(constitutive

mut.)

iS

(super--reperessible)?

Negative—inducible

operon

I

(來源：不詳)bindingbetween

labeledlacO-DNA

with

32pand

added

increasingamountsof

lac

repressor.(Source:Cohn.

Journal

of

Molecular

Biology,

Vol.

34:366.1968)Lac

or

IPTG

lactose

analog

is

inducer

of

Lac

operonIPTG;異丙基－β

－D－硫代半乳糖苷，自己不被分解的強誘導物（安慰誘導物）O

gene

(operator)

cis-action

factormRNAstartpointunwindingRNA

polymerasebindingRepressorbindingObstruction?Competition?(來源：不詳)O

gene

(operator)

cis-action

factorSymmetrical

seq.

of

21

bp

&

G/C

as

axisT

can

be

crosslinked

to

repressor(bindingsite)Methylationenhancedby

repressorProtectedby

repressoragainstmethylation(來源：不詳)O

gene

(operator)

cis-action

factormRNAstartpointunwindingRNA

polymerasebindingRepressorbindingO

&

Poverlap

repressor&

RNApolymerase

bindat

sites

that

overlap

around

the

start

point

of

Lac

operonrepressor&

RNA

polymerase

對重疊位點競爭O

gene

(operator)

cis-action

factorObstructionO

gene

(operator)

cis-action

factorO+

—

mut.

OC

occur

frequently

at

left

site

of

axis

(?)調(diào)控機理Inducer(lactose)

與repressor特異結(jié)合tetramer

變構(gòu)

特異結(jié)合力下降1000X作用于

O

位點上的repressor

變構(gòu)

脫離O位作用于游離的repressoroperononrepressortetramer與operator發(fā)生特異結(jié)合operon

off變構(gòu)

失去結(jié)合于O位的能力w.t.

(I+

O+

P+)

誘導型add

inducer

operon

on

no

inducer

operon

offOC失去與repressor特異結(jié)合的能力OC

mut.

(I+

OC

P+)

constitutive

mut.

（組成型）The

Oc

lac

operatorbinds

repressorwith

loweraffinitythan

does

the

wild-type

operator,

Oc

required

a

higherconcentrationof

repressorto

achieve

full

binding.wild-typeoperator(O+)operator-constitutivemutation(Oc)control入Ф80DNA,(Source:Molecular

Biology(2002),Robert

F.Weaver,Page179）iC

mut.

(iC

O+P+)

constitutive

mut.

(組成型)iC

gene產(chǎn)物repressor喪失與O位點結(jié)合的能力iS

mut.

(iS

O+P+)

super-repression

mut.

(超阻型)iS

gene

產(chǎn)物repessor不能與inducer結(jié)合等位基因間的顯隱關系icp

oZYAicI

+ictetramerNon

bindingrepressor

tetramertetramertetramerLactose變構(gòu)iC

gene產(chǎn)物repressor喪失與O位點結(jié)合的能力等位基因間的顯隱關系I+/IC

I+

>

iC(Source:Molecular

Biology(2002),Robert

F.Weaver,Page177）p

o

iSmutrepressortetramerlactoseiS

gene

產(chǎn)物repessor不能與inducer結(jié)合

IS/I+iS

>

I+

iS

>

iC

等位基因間的顯隱關系I+

(Source:Molecular

Biology(2002),Robert

F.Weaver,Page178）OC失去與repressor

特異結(jié)合的能力

OC

＞

O+

cis-dominantI+OCZYAlactoseI+

O+等位基因間的顯隱關系mRNA

mRNAcis-dominantThe

ability

of

a

site

(cis-factor)

tocontrol

adjacent

gene

irrespective

ofthe

presence

in

the

cell

of

other

allelesof

the

site.cis

acting

factor

對與其緊密連鎖基因的控制效應不受其等位基因的影響。Inactive

expressoractive

expressoroperon

off?Positive—inducible

operon

(多為分解酶類)

I

inactive

expressor

(apoinducer誘導蛋白)induceractive

expressor

binding

on

front

Psitew.t.

(I+O+P+)

誘導型iSmut.

超阻突變

(super-repression)expressorcan

not

be

activated

by

induceroperon

offI+

>

iS

(來源：不詳)激活RNApolymerase啟動Lactose

Lac

operon

openbut

no

transcripts

J.

Monod

Why?diauxy

e.g.

cAMP

control

(universal

controllingsystem)

(Source:Molecular

Biology(2002),Robert

F.Weaver,Page173）

E.coliGlucoseJ.

Monod(Source:Molecular

Biology(2002),Robert

F.Weaver,Page173）ATPppp

cAcAMPpcAp

c

A5‘-AMPI

(cap

gene)(cAMPacceptorprotein)(or

catabilitegene

activatorprotein)cAMPase(-)phosphodiesterase

(+)+GlucoseLac.Operon

I

P

OStimulation

of

β-galactosidasesynthesis

by

cAMPwithwild-typeand

mutant

CAP

(Source:Pastan

1970

P.

N.

A.

S.

480-487,June

66

(2))CAP-cAMPcomplexis

important

for

lacoperon

transcriptionBut

too

much

cAMP

obviouslyinterferedwith

β-galactosidasesynthesis.And

cAMP

has

manyeffects,

some

may

indirectlyinhibit

some

step

in

expressionof

the

LacZ

gene

in

vitroNullNothing

(Source:Molecular

Biology(2002),Robert

F.Weaver,Page184）The

lac

control

region.just

upstream

of

the

operator,contains

the

activatorsite

(orCAPbinding

site)

AR1

AR2

AR1

AR2

(Source:Molecular

Biology(2002),Robert

F.Weaver,Page184）CAPplus

cAMPallow

formation

of

an

open

promoter

complexThe

CAP-cAMPdimer

binds

to

its

activatorsite

on

the

DNA,and

the

α-CTD

interacts

with

a

specific

site

on

the

CAPproteinThis

strengthens

binding

between

polymerase

and

promoter.,Hypothesis

forCAP-cAMPactivation

of

lac(Source:Busby,

S.

and

R.H.

Ebright

Cell

79:742,1994)cAMP—CAP具有廣泛生理效應的正控制系統(tǒng)存在于多種基因表達調(diào)控體系中Lac

operon

expression

cAMP(

Positive-inducible)

lactose(

Negative-inducible

)d)

基因表達調(diào)控的綜合實例

λ

phage

發(fā)育階段選擇調(diào)控的分子生物學

Histidine

Utilization

operon

(Hut)e.g.

1

Histidine

Utilization

operon

(Hut)

a)

Histidine

代謝過程Histidine尿苷酸鹽咪唑丙酮酸鹽HistitaseUrocanaseIPAhydrolase

亞氨谷氨酸鹽FGAhydrolaseGlutamate

GluPIGRepressorCPOOUHGPOUHCNo

Histidine

P

O

IHistidine

present

b)

operon

type

&

structure

Negative—inducibleoperon

(regulon)

by

C

gene

(repressor)

Histidine

as

inducer(來源：分子生物學（2007），鄭用璉，第226頁)Histidine

presentRepressor

PPII

GGP

POOOOUU

HH

CCCAP-cAMP

Positive—inducibleoperon

by

CAP—cAMP(來源：分子生物學（2007），鄭用璉，第226頁)Histidine

presentRepressor

PPII

GGP

POOOOUU

HH

CC活化的谷氨酰胺合成酶GS(或NR-pi)Positive—inducibleoperon

by

GS

(Glutamine

synthetase)

NR-pi

(phosphorylated

Nitrogen

regulator)(來源：分子生物學（2007），鄭用璉，第226頁)c)

GS

positive—induciblecontrol

model(active)NH3NR1

+AMP(inactive)NR1-Pi

NO3－(active)PhosphorylatedNitrate

Reductase

PII(ATPase‘s

regulatoryprotein)PII-(UMP)4Uridilate-PIIStimulated

byα-ketoglutarate

Stimulated

by

Glutamine(Gln)UTase4

UMP

H2O(UTP)4

(ppi)4UTase

NR1(inactive)ATPADP

NR1-Pi

Binds

to

upstream

site

of

operon

GStranscriptionα-ketoglutarate

NR1-Pi

(GS)

Positive

controlHut

0peronNH3

+

α-ketoglutarate

When

N

starvedGlu

+

NH3

+ATPGln

+ADP

+

Pi

GlnGS

idling

reactionoperon

type

&

structureNegative—inducibleoperon

(regulon)by

C

gene

(repressor)Positive—inducibleoperonby

GS(Glutamine

synthetase)NR-pi

(phosphorylatedNitrogen

Regulator)CAP—cAMPe.g.

2λ

phage

發(fā)育階段選擇調(diào)控的分子生物學--λ

phage

溶原(lysogenic)和裂解(lytic)的發(fā)育途徑--λ

phage發(fā)育途徑選擇調(diào)控區(qū)的基因組成λ

phage發(fā)育途徑選擇調(diào)控的分子基礎Lytic

versus

lysogenic

infection

by

phage

入(來源：分子生物學（2007），鄭用璉，第233頁)Head

TailPROR

CRO

tR1

CII

O

P

Q

S

RtL2

int

Pint

Xis

CIII

tL1

N

OLPL

CIPRMP

REcoscosCis-

factorTrans-factorForreplicationlysogenyλ

phage發(fā)育途徑選擇調(diào)控區(qū)的基因組成

Late

transcriptioncontrollysisrecombinationlysisλphage

developing

stageImmediate

early

stage;N

&

Cro

genes

transcriptionfrom

PL&

PRDelayedearly

stage;N-p

required

for

anti-terminationat

NuTL

&

NuTRLate

stage;Q-p

requiredFrom

PR‘(來源：分子生物學（2007），鄭用璉，第236頁)λ

phage發(fā)育階段和調(diào)控區(qū)lateDelayed

early

EarlylateDelayed

earlyEarlyHead

TailPROR

CRO

tR1

CII

O

P

Q

S

RtL2

int

Pint

Xis

CIII

tL1

N

OLPL

CIPRMP

REcoscosPR’CI

cI可建立溶原，但不易維持λ

phage發(fā)育途徑選擇調(diào)控區(qū)的基因組成C

gene

（與溶原直接相關）

λ

host

lysogenic

turbid

colony

C

gene

mut.

lytic

Clear

plaqueCI-p

26kdas

repressorbinding

on

PROR,

PLOL

（負控制）as

expresser

for

CI

self

（

正控制）CII

cII不易建立溶原，一旦建立溶原后，

依靠CI-p可保持溶原

CII-p

as

expresser

啟動PRE

轉(zhuǎn)錄CI

geneCIII

cIII

參與溶原建立過程

但對溶原化作用不大

CIII-p

as

expresser

啟動PRE

轉(zhuǎn)錄

CI

geneOR3OR2OR1OL3OL2OL1PRCROCICI

PRMG/C

as

axisIR

seq

of

17

bpOR1,

OR2,

OR3

序列的差異，決定了CI-p,

CRO-p與其的結(jié)合力For

CI-p

(repressor)OR1

＞

OR2

＝

OR3For

CRO-p

(repressor)OR1

＝OR2

＜OR3Operator

(cis-factor)OR

(OR1,OR2,

OR3),

OL

(OL1,OL2,

OL3)(來源：分子生物學（2007），鄭用璉，第235頁)Monomer(200)

Dimer(50)結(jié)合于O位點PRMCI-p/CRO-p的標準濃度

3

mM

為1標準濃度單位

genotypeOR3

OR2

OR1

OR3CI-p

25OR2

PRCI

2OR1

CI

1OR3

OR2

OR1188

OR3CRO-p

OR2CRO

OR1CRORegulator

(trans-factor)

acidicprotein

236

aa,

26kd

C-end

dimerization

domain

N-end

DNA

binding

domain

(Control

of

Repressorand

Other

things)66aa

(3

helix

&

3

sheets)Dimerizationdomain

&

DNA

bimnding

domain

CI-pCRO-pN-pAnti-termination

proteinfor

delayed

early

stageQ-pAnti-termination

proteinfor

late

stagePRE

(Promoter

forRepressorEstablishment

PE

(Promoter

for

lysogenic-Establishment

Locatedbetween

CROCIIPromoter

Head

TailtL2

int

Pint

Xis

CIII

tL1

N

OLPL

CIPRMP

REcosStrong

promoterPositive

controlsite

with

CII-p,CIII-pTranscriptionCI

gene

&

anti-sense

CRO

RNA

PROR

CRO

tR1

CII

O

P

Q

S

R

cosPRM

(Promoter

for

RepressorMaintenance)

PM

(Promoter

for

lysogenic-Maintenance)

Locatedbetween

OR2—OR3Weak

promoter

(1/7

~

1/8

of

PRE)Positive

controlsite

with

CI-pNegative

control

site

with

CI-p

&

CRO-pTranscriptionCI

gene

PROR

CRO

tR1

CII

O

P

Q

S

RtL2

int

Pint

Xis

CIII

tL1

N

OLPL

CIPRMP

RE

Promoter

Head

TailcoscosHead

TailPROR

CRO

tR1

CII

O

P

Q

S

RtL2

int

Pint

Xis

CIII

tL1

N

OLPL

CIPRMP

REcoscosPromoter

PR

(Promoter

on

Right)

Locatedbetween

OR1

—

OR2

Negative

control

site

with

CI-p

&

CRO-p

TranscriptionCRO,

CII,

O,

P,

Q,

S,

R,

H,

T

genes

PR’Promoter

PL

(Promoter

on

left)

Locatedbetween

OL1

—

OL2

Negative

control

site

with

CI-p

&

Cro-p

TranscriptionN,

CIII

genesHead

TailPROR

CRO

tR1

CII

O

P

Q

S

RtL2

int

Pint

Xis

CIII

tL1

N

OLPL

CIPRMP

REcoscosPint

(Promoter

for

Integration)

Locatedon

the

downstreamof

CIII

Positive

controlwith

CII-p

&

CIII-pPromoterHead

TailPROR

CRO

tR1

CII

O

P

Q

S

RtL2

int

Pint

Xis

CIII

tL1

N

OLPL

CIPRMP

REcoscosEarly

stage;

RNApol

RNApolPRORPLOL

Regulation

modeltranscriptionCRO-p

Stop

at

TR

1transcriptionN-p

Stop

at

TL1cosPRMcos

Early

stageHead

TailtL2

int

Pint

Xis

CIII

tL1

N

OLPL

CI

Early

N-P

Anti-termination

P

REDelayed

early

stage

CRO-p

EarlyPROR

CRO

tR1

CII

O

P

Q

S

RNutRNutL(a)

NusAbinds

to

polymerase,

and

Nbinds

to

both

NusAand

box

B

of

the

nut

siteregion,

creating

a

loop

in

the

growing

RNA.The

antitermination

is

caused

by

inhibitingterminatorhairpin

formation(來源：分子生物學（2007），鄭用璉，第237頁)Head

TailPROR

CRO

tR1

CII

O

P

Q

S

RtL2

int

Pint

Xis

CIII

tL1

N

OLPL

CIPRMP

REcosDelayed

early

stageCII-p

CI

+

anti-senseCRO-mRNACIII-pCRO-pinteractionbetween

CII

and

two

early

入

promoters(Source:Ptashne.

Nature

304

(1983)p.

705)1)

none

CII2)

10

pmol

of

CII3)

18

pmol

of

CII4)

90

pmol

of

CIIThe

CII

footprint

in

promoterincludes

the

-35

box

(positive

control

site)PLOLCIPRMPRECI

mRNA

+

anti

RNAof

CROCI-PPLOLCIPRMPRECI

mRNA

+

anti

RNAof

CRO

PR

CRO

PR

CROCI-PEstablishesMaintenanceLysogenicCIPLOL

PRORPRM(來源：不詳)Maintaininglysogeny(來源：分子生物學（2007），鄭用璉，第239頁)The

repressorclearly

inhibited

cro

transcriptionat

lowconcentration,but

inhibited

cl

transcription

at

high

concentration.(Ptashne

Meyer

et

al.

Repressor

turns

off

transcription

of

its

own

gene.P/VAS

72

(Dec

1975)Increasingconcentrations

ofrepressorCI.check

the

cl

and

crotranscripts,(來源：分子生物學（2007），鄭用璉，第236頁)OR3

OR2PRMOL3OL2OL1OR3

OR2PRMOR1

OR1PR

OL3OL2OL1PRCICIDelayed

early

stagePositive

anti-senseCRO-mRNAEstablishes&

MaintenanceLysogenicPRMPintCII-prepression

CI-pOR1,OR2PR

offPositivetranscriptionPRORPLOLtranscriptionCRO-p

&

CII-ptranscriptionN-p

&

CIII-pRNApolCI-pPREInt-pRNApolPLOLN-p

&

CIII-p

PositiveOR3

OR2

OR1PRMCROPRCRO

PROR3

OR2

OR1CIPRM

CIRec.ADelayedearlystageUV

or

木瓜蛋白酶CI-Pis

bound

to

OR

(and

OL)and

cl

is

being

activelytranscribedfrom

the

PRMpromoter.The

RecAco-proteaseunmasksa

proteaseactivityin

the

repressor,soit

can

cleaveitself.The

severedrepressorfallsoff

the

operator,allowingpolymeraseto

bind

toPR

and

transcribecro.

Lysogenyis

broken.CROCI

OR2

OR1PRMOR3

OR2

OR1PRMCRO

OR3PRUV

or

木瓜蛋白酶

CIRec.ALate

stage

PRCI-p

monomerU.V

or

木瓜蛋白酶…RNApol.

cleavage

SOS動態(tài)平衡破壞

PR

onQ,

P,

R,

S…..轉(zhuǎn)錄OR3+

CRO-p轉(zhuǎn)錄PRM

offLate

stage

Head

&tail

genes

positive

Q-p

Lytic

stageLate

stageQ-p

binding

to

qut

site

（overlapping16-17bp

with

PR‘）+Q:

Q識別暫停復合體并與qut位點結(jié)合，然后Q結(jié)合到RNA聚合酶上，忽略終止子，繼續(xù)轉(zhuǎn)錄晚期基因。R-S-H-T-Q:

RNApol到達Pausesite后暫停數(shù)分鐘，

越過暫停

位點，只轉(zhuǎn)錄到終止子后便放棄晚期基因的轉(zhuǎn)錄。U.V

or

木瓜蛋白酶…cleavageLate

stageLytic

stage誘導裂解

鼠逃

自身

即將沉沒的船》？

SOS響應溶源菌遭受DNA損傷的信號

幫助λ通過誘導裂解遠離不利環(huán)境《老

對λ

離

有什么進化優(yōu)勢具有選擇

lytic

趨勢自然選擇形成；

λhostRNApol.

binding

PROR

early

stageCRO-p

binding

OR3CRO-p

off

PRMLate

stage

other

controlling

system

for

Lytic

way

selection

The

purpose

of

λ

phage

infection

E.coli

Lysis

the

host

of

E.coli如果cI先表達則建立溶源態(tài)，如果cro勝出則被侵染的細菌會裂解。如果cI基因產(chǎn)生足夠多的阻抑物，那么這些蛋

白會結(jié)合到OR和OL上阻止早期基因的進一步

轉(zhuǎn)錄如果產(chǎn)生了足夠的CRO蛋白，它會阻止cI基因的轉(zhuǎn)錄從而阻礙溶源態(tài)的建立。什么因素決定是cI還是cro在競爭中勝出呢？CII的濃度CII的濃度越高，就傾向于形成溶源態(tài)控制著CII的濃度細胞內(nèi)蛋白酶的濃度（HFL）培養(yǎng)營養(yǎng)環(huán)境因素為什么λ噬菌體又要選擇溶

原途徑作為自己生活周期的一種方式呢？選擇溶原途徑又具有什么生物學意義呢？HFLgeneHFL-p?

HFL

gene

control

of

host

(high

frequencylysogenesis)When

[C]

starvedLysogenicmut.

hfldegradation

CII-plytic

HFL

gene

off

(籌集糧草)degradationCII-pPositive.CII的濃度越高，就傾向于形成溶源態(tài)

控制著CII的濃度進入

溶源CII濃

度高細胞內(nèi)蛋白酶的濃度（HFL）

蛋白酶

濃度低

培養(yǎng)營養(yǎng)環(huán)境因素

碳源

不足?

MOI

≥10

(Multiplicity

of

infection)

CI-pLysogenicλ

/E.coli

高

CII-p

monomer

多

但不穩(wěn)定激活PRE

CIII－p抑制HFL蛋

白酶對CII-p的降解CII-pdimer

多穩(wěn)定sibTL2IntPint

xisCIII

TL1

NPLOL

mRNA

degradated

by

3’—5’exonuclease

from

sib

to

xis

(inclusion

int)sib

site

negative

control

Int

gene

by

sib

(retro-regulation)此時λ

并未決定進入溶原時期，不需int-p

抗終止蛋白N-p

sibRNAaseIII?

Int

gene

and

retro-regulationof

sib

sitelysogenic

wayIntegrataseTL2Int

Pint

xisCIIITL1

NPLOLpol

polWhen

lysogenic

way

be

selected

Xis

gene

RNA

RNA

RNApol

CII-p

Int

mRNANo

XIS-p

(Pint

located

in

the

region

of

xis)sibTL2IntPintxisCIII

TL1

NPLOLattP

POP‘λ

的整合與切除

Int

(Integrase)特異性識別

POP?,

BOB?Xis

(Excisionase)特異性識別BOP?

,

POB?BOP?XisSib

POB?PLOLExcisionaseattPof

λp

Op‘a(chǎn)ttB

of

E.coli

B

O

B‘PLOLCRO-p

N-p

sib

retro-regulationCRO-p

precedenceover

CII-p

HFL-p

Q-pCI-p

Pos.

PRMCII-p,CIII-ppos.

PRE

antisenseCRO

mRNA

CI-p

gene

expressionCI-p

U.V

degradation

CI-pconclusionLate

S.Lytic

S.Delay

early

S.λHostEarly

S.CII-p

pos.

PintNeg.

PR

RNApol

+

PR

MaintenanceMOI≥10,CRO-p

neg.

PRM

C

starved

PL

XisEstablishementLysogenic

U.V

degradation

CI-p

6.1.2.

Attenuator

controla)

discovery●E.coli

trp

synthetase

operon(

C.

Yanofsky

stanford

Univ.

)

Negative-repressible

operon70-fold

lower

than

fully

de-repressedIPOleading

seq.EDCBAtrp+●1968.Imamato

Lab.IPOleading

seq.EDCBA

Low

trpWhen

trp

level

is

low，RNApol

moves

but

falls

off

in

the

L.S.and

the

transcription

of

the

structure

genes

is

blocked.+the

complex

can

not

bind

with

the

O

site●1975

Imamato

Lab.E.coli

trp-AARS

(t.s)

30℃42℃trpEtrpEProteinsynthesisTrptRNAtrp30

℃42

℃trp-tRNAtrpNothing

t.s.(42℃)

t.s.(30℃)w.t.(30℃)w.t.(42℃)

Low

trp

，

operon

opentrpEWhy?

w.t.AARS

t.s.mut.?

t.s.(42℃)AARS

inactive

no

trp-tRNAtrpsynthesisRNApol

move

past

L.S

transcript

of

trp

synthetase

RNA(E

enzyme

level

is

high）?

t.s.

(30℃),

w.t.(30℃),

(42℃)AARS

active

synthesize

trp-tRNAtrpRNApol

break

off

at

L.S.

no

transcriptionof

trp

synthetase

RNA（

E

enzyme

level

is

low）Conclusion:When

trp

and/or

trp-tRNAtrpexist

in

cells,there

is

no

synthesis

of

trp(Fine-tuned

control)

t.s.(42℃)

noAARS

and

trp-tRNAtrp,but

take

for

no

trp

by

error,（initiate

the

transcription

of

trp

synthetase

RNA)trp-tRNAtrp

is

the

main

factorleading

to

RNApol.

fallingoff

at

L.S.●Fine-tuned

regulationwithin

the

leading

sequenceIpoL.S.E

D

C

B

AgenotypeI+iCTrp

Δ

ED

0.171.32

11.8100When

trp

is

rich,B

activitydetected

I+;

operon

off

low

E

activity

i

C;

operon

on

high

E

activity(~70X)The

differenceof

E

activityof

Δ

ED

&

(~10X)

resultsfrom

the

L.

S.

Controlsthe

operon

over

a

700-fold,

range

from

fullyinactiveto

fullyactive0.17100??

leading

seq

with

cis-acting

domain

Partialdiploidtest（trprich

）GenotypeE

activity

AactivityL.S.

withoutcomplementaryeffect.L.S.

is

a

cis

factorwith

attenuationeffect.Cis-dominantFtrpiCL.S.EDCBA1.414.8trp

iCΔ

AL.S.EDCB1.10L.S.

trp

iCΔ

Etrp

iCΔ

L-C

trp

iCΔ

L-CDCB

A

BA

BA0

0

2.516.5

94

102p-

trp

iCΔ

A

p

L.S.

E

D

C

Bb)

The

primary

structure

of

trp

operon

RNA?

60~68—75~83&

110~121—126~134(palindromicseq.)

with

poly

(U)?

27—68

base

ORFof

14aa?

140

base

RNAalmost

binding

with

protein

(translatable

seq.)?

trp

high

frequency

with

1/7

in

14

aa(來源：分子生物學（2007），鄭用璉，第228頁)Peptides

of

L.

S.

for

a

few

Operons

encoding

enzymes

for

amino

acidsynthesis

where

transcriptionattenuation

occurs.(7/16)(7/16)(8/15)What

is

meaning

?(來源：分子生物學（2007），鄭用璉，第229頁)

The

expression

of

trp

operon

under

the

a.a

starvationE.colitrpiC

u-cultures

in

the

medium

with

9

differenta.a.

starved,

Respectively.

Add

H3-U，detect

trp

operon

mRNA?

Trp

codon

andorderplay

animportant

role

onthe

expression

ofthe

operon?

butArg

isimportant

likewise!

Why?H3-U

mRNA

Met

Ile

Leu

Gly

Trp

Arg

Thr

His

Proc)

The

secondary

structure

of

the

140

Nt

RNA

?

E.coli

DNAHpaII含L.S.

570

dNt

DNAfrag.

In

vitro

transcription

140

Nt

RNA

G

N

RnaseT1(

P

P

)Denaturalization/Non-denaturalization

PAGE

&

Northern

blottingElimination

of

the2nd

structureAC

B14071

694332

25

18AC

B

no

Ureawith

Urea

RecycleA,

B,

C

bands

AUrea

gel

B

C

Conclusion:Aband（140Nt）70thNt

is

G,

locates

in

the

single

strandB

band（108-140Nt）no

G

exposes

in

the

single

strandC

band（52-94Nt）52th--70th--94th

is

a

fragment

ofA

band‘sG52G94140AG70G108BG52G94G70C1-2

/

3-4Alternative

pairing

scheme

of

3

forms

for

threesequence

in

the

trp

mRNAleader.

2-3(來源：分子生物學（2007），鄭用璉，第228頁)Trp(Arg)

starved

Arg

60-

-70-

-trp

trp55-

-

58-

-

65-

-68-

-

Non-starved

UGA69

-

-

79

-

-2-33-4

Rho-independent

T.(來源：不詳)14aaPARibosome

pass

through

Trp

codon

(來源：不詳)9aaPARibosome

pausing

at

Trp

codon

(來源：不詳)e)

The

biological

significance

ofAttenuation

controlWhen

trp

level

is

lowrepressoris

not

enough

to

close

operonIf

anly

trp-tRNAtrp

exist

in

cells,

Attenuator

will

interrupt

the

RNApol.

transcription

in

the

L.S.When

trp

level

is

highlittle

RNApol.

Pass

through

the

O

site+

trp

Protein

synthesis

trp-tRNAtrpI

po

L.S.

E

D

C

BA

tRNAtrp+AARS

trpTrp

synthetase

opronThe

fine-tuned

regulation

in

the

ProkaryotesEnhance

the

adaptability

of

prokaryotesto

the

environmentbiologicalsignificanceThe

mechanism

ofAttenuation

controlYanofsky

Charles

.

1

981.

Nature.

289;751-758Control

the

transcription

of

the

operon

finely

via

the

translation

of

the

shortpeptide

coded

in

the

L.S.第6章

基因表達調(diào)控(Controlling

of

the

Gene

Expression)(Source:Pique,Michaeland

Peter

E.Wright,Dept.(cover

photo,science

245

(11Aug

1989).))6.2

post-transcriptional

level

control

pre-RNA

processing(for

Eukaryots

only)

Anti

sense

RNA

and

RNA

interference

(RNAi

)廣泛存在于原核生物（E.coli）

真核生物（plant-mammalian）基因沉默

位置效應（positioneffect）

整合位點轉(zhuǎn)錄水平的基因沉默（transcriptional

genesilencing,

TGS）

啟動子甲基化或?qū)牖虍惾旧|(zhì)化轉(zhuǎn)錄后水平的沉默(post-transcriptional

genesilencing,

PTGS)低滲透壓

高滲透壓OmpCRNApol

OmpC

mRNAompC

OmpF外膜蛋白

OmpF

micFRNA

anti-sense

RNA

control

protein

translation

(Source:1983.

Miruno

&

Simons

)

高滲透壓

6S

174

base(mRNA-interfering

complementary

RNA)

OmpF

mRNAmicRNA

(anti-sense

RNA)

binding5’-end

of

mRNA(S.D.seq.

&

AUG.)

micF-RNA

Initiationcodon

OmpF-mRNA(來源：不詳)

遺傳特點anti-sense

gene

of

CHS

(

Chalcone

synthesis

gene

)white

flowerred

flower×red

flower

dominance

/recessive?non

complete

dominancestable

inheritanceD.S.

RNA

unstable

and

rapidly

degradated"for

their

discovery

of

RNAinterference

-

gene

silencing

bydouble-stranded

RNA".NP2006

CraigC.Mello馬薩諸塞州醫(yī)學院

AndrewZ.Fire,斯坦福大學醫(yī)學院6.2.2.2RNAinterference(RNAi

)的發(fā)現(xiàn)與證實RNA

interference

(RNAi

)的發(fā)現(xiàn)與證實

1990年，RichJorgensen等人發(fā)現(xiàn)

苯基苯乙烯酮合成酶基因

矮牽?；伾囫g全白色素的合成不是被增強了，而是被關閉了Su

Guo

1995