分子生物學第五章課件

第五章

蛋

白

質(zhì)

翻

譯來源：不詳5.1.

基本元件5.2.

Genetic

Code5.3.

peptide

synthesize5.4.

保證peptide準確翻譯的機制5.5.

Central

Dogma

的發(fā)展5.1.基

本

概

念●

codon;universal

triplex

codon

two

of

three

reading

codon

paracodon

codon

in

codon●

codon

degeneracy;wobble

hypothesis

isoacceptor●

codon

usage

(codon

bias)●

mechanism

of

accurate

translation

initiation,

loading,

elongation,

proofreading5.2.

基

本

元

件(Source:IrvingGeis/Peter

Arnold,Inc.)5.1.1.

tRNA●

mini

RNA,

4s,

(70-80

Nt)●

tRNA

phe,

77Nt

cloverleaf

form

(1964

HollyR.)●

5

arms

&

4

loops●

Nt

more

modified

by

methylationCapping

Cap

0:

m7GpppXpYp

Cap

1:

m7GpppXmpYp

Cap

2:

m7GpppXmpYmp

Help

the

splicing

of

the

first

intronPre-RNA

tailing

A

poly(A)

tail

(50-200±)

be

added

at

-20

Nt

±

tailing

signal

(AAUAAA)

from

3’-end

of

Pre-RNA

Specific

endonulcease

recognizes

AAUAAA

and

the

following

GUGUGUG,

cuts

within

the

sequence,

adding

poly(A)s

at

3’-end5-1tRNA

mini

RNA,

4s,

(70-80

Nt)

Nt

more

modified

by

methylation

tRNA

phe,

77Nt

cloverleaf

form

Aa

accept

arm,

DHU

loop

(contact

with

AARS),

anti-

codon

loop,

TΨC

loop

(contact

with

5S

rRNA),

extra

loop

Paracodon:a

numberof

Nts,

on

tRNA,

contact

with

AARSChapter

5

Protein

translationtRNA的”L”三維結(jié)構(gòu)(來源：分子生物學（2007），鄭用璉，第180頁)“L”結(jié)構(gòu)域的功能aa

accept

arm

位于“L”的一端，契合于核糖體的肽基轉(zhuǎn)移酶結(jié)合位點

P

A,

以利肽鍵的形成anti-codon

arm

位于”L”另一端，與結(jié)合在核糖體小亞基上的codonof

mRNA配對aa’-tRNAaa(來源：不詳)“L”結(jié)構(gòu)中堿基堆積力大使其拓撲結(jié)構(gòu)趨于穩(wěn)定wobble

base位于“L”結(jié)構(gòu)末端堆積力小自由度大使堿基配對搖擺

TΨ

C

loop

&

DHU

loop

位于“L”兩臂的交界處，

利于“L”結(jié)構(gòu)的穩(wěn)定Source:Quigley,G.J,Structural

domains

of

transfer

RNA

moleculars,Science

194:197?

have

GC

content

of

60%

&

Rich

methylation?

each

cell

contains

from

several

hundred

to

over20,000

copies

of

rDNA

gene?

rRNA

synthesized

in

nucleolus

and

was

stimulatedby

low

ionic

strength

&

Mg+2Ribosomal

genes

(rDNA)

are

differentin

several

ways

from

other

nuclear

gene5.1.2.

rRNA●●Prokaryote

23s,

16s,

5s

/

Eukaryote

28s-5.8s,18s,

5sRich

methylation

(m2U,

m3A,

m3U,

m26A(二甲基)…)

(來源：不詳)●

23s

rRNA

6

domains有的與對抗生素的抗性有關(guān)2660±Ntregion

α

-I

loop

(alpha

Sarcin)binding

with

complex

of

aa-tRNAaa~(EF)-Tu~GTP(引起核糖體變構(gòu)??！)G2661

C

,

aa-tRNAaa

intoAsite

go

downG2252,

G2253雙突變?yōu)镃,將對轉(zhuǎn)肽酶的活性產(chǎn)生抑制

In

Euk.

3?-end

of

18s

rRNA

與原核生物高度相似,

但無與

S.D.seq.互補的保守序列在

mRNA的AUG上游存在CCACC核糖體scanningseq

成為核糖體識別第一個AUG的信號AMEAMECCUGCGGUUGGAUGACCUCCUUBacterial

16S18ＳMammalianAMEAMECCUGCGGAAGGAUGAUUA

高度相似●

In

Eukaryotemono-cistron

leading

seq.5?m7Gppp

CCACCA-3A1U2G3G4—核糖體小亞基掃描AUG至關(guān)準確翻譯

的信號序列But

mRNA

of

chloroplastshows

similarities

to

prokaryote

type1;

S-D

seq.

with

greatersecondary

structurein

L.

S.

type2;

richAU

with

littlesecondary

structurein

L.

S.

polycistron5.2.

Genetic

CodeSource:Oscar

Miller/SPL/Photo

Researchers,Inc

In

vitro

Poly(U)

Poly(C)

Poly(A)

Poly(G)

Butpoly(UCUCUC…)

UCU/CUC

poly(Phe)

peptidepoly(Pro)peptidepoly(Lys)

peptidepoly(Gly)

peptidepoly(Ser-Leu-Ser-Leu…)

Ser/Leu

?密碼子的破譯

(1968.nobel

prize)

Marshall

Nirenberg

(1961)M.

Nirenberg

&

P.

Leder

(1964.

Science

145;1399)In

vitro

UCU(trinucleotides)Ser-C14,

Leu,

Lys,Arg,…Ser,

Leu-C14,

Lys,Arg,…Ser,

Leu,

Lys-C14,Arg,…

……

tRNAaa

Ribosome

Nitrocellulose

filterSer-C14….Leu-C14….Lys-C14….Gly-C14….(來源：分子生物學（2007），鄭用璉，第188頁)發(fā)生終止突變的原氨基酸Trp

(UGG)UAC

Gln

:

CAG,

CAA

Glu

:

GAG,

GAA證明：終止突變密碼為UAG

(amber

琥珀突變)UAA(ocher

赭色突變)UGA(opal

蛋白石突變)?!Stop

codon

的證實

aa

and

codon

in

back

mutant

Ser:

UCG,

UCC,

UCA,

UCU,

AGU,AGC

Leu

:

UUG,

UUA,

CUU,

CUC,

CUA,

CUGUAGUAGTyr

:

UAU,

UAGLys

:AAG,

AAAUAGUAGUAGＸＹＺＵＧＧ(來源：分子生物學（2007），鄭用璉，第190頁)5.2.2.

Degeneracy

of

codon

(密碼子的簡并現(xiàn)象)a)

簡并現(xiàn)象的概念；一種氨基酸受2個以上codon編碼的遺傳現(xiàn)象編碼一種aa的4個codon間,僅3rd

Nt

不同,稱為

codon

family例；Ser(6

codons)

1

codon

family

&

2

extra

codons5?3?

A

Ab)

簡并現(xiàn)象的機理；●

●Isoacceptor;負載同一氨基酸，但識別不同密碼子

的不同tRNA

Wobble

hypothesis;GCGGCUUCU5?3?5?3?

AtRNA3

isoacceptors1

codon

family

2

extra

codons

反密碼子

:

密碼子

在一定范圍內(nèi)的可選擇配對現(xiàn)象

1th(Nt34)

:

3

rd-NtmRNA5?CGUCGCCGACGGAGAAGG3?

wobble?!簡并現(xiàn)象的機理；●Isoacceptor;

負載同一氨基酸，但識別不同密碼子

的不同tRNA負載同一氨基酸，識別相同密碼子的不同tRNA？！Tyr

codon:UACAUGAUG識別UAC

Codon負載Try的tRNA有兩個，但結(jié)構(gòu)向差較大。

Y

YtRNAmeti

&

tRNAmete

;

tRNAmetf

&

tRNAmetm存在明顯的結(jié)構(gòu)差異5?5?3?No

base

pairingtRNAmetftRNAmetmM

3?M

G-C

rich

G-C

rich(來源：分子生物學（2007），鄭用璉，第196頁)CGCGCG

CGU

1

3

(來源：分子生物學（2007），鄭用璉，第193頁)●

Wobble

base的搖擺配對原則GUG（val）的第一Nt會以較低頻率與tRNAmetf反密碼子(CAU)發(fā)生“搖擺”配對，而作為起始密碼.(E.coli

GUG

/

AUG

=1/30)(Source:Molecular

Biology(2002),Robert

F.Weaver,Page570）mRNA(1GUG)（val）作為起始密碼.

與tRNAfmet的反密碼子(CA3U)配對，不是真正意義上的”搖擺”.由于tRNAmet

f中反密碼子下游第一個Nt(37)為未修飾的A，而其他tRNA第37個Nt幾乎為較大的烷化修飾的Nt例如tRNAmet

m第37個Nt為t6A(N6-蘇氨酸羰酸腺苷)tRNAmeti

&

tRNAmete

;

tRNAmetf

&

tRNAmetm存在明顯的結(jié)構(gòu)差異5?5?3?No

base

pairingtRNAmetftRNAmetmM

3?M

G-C

rich

G-C

rich

N6-蘇氨酸

羰酸腺苷(來源：分子生物學（2007），鄭用璉，第196頁)意味著反密碼子邊序堿基修飾對限制錯讀的機制AUG

CA3UA（37）mRNAtRNAmetftRNAmetm

1GUGCA3UA（37）

AUG

CAUt6A（37）堿基搖擺配對的方式

C

G

U

GThio-U

G

C

I

U

A

Thio-U

AU

Iss

A

I(Source:Molecular

Biology(2002),Robert

F.Weaver,Page570）●Wobble

base搖擺配對的機理

tRNA的拓撲空間結(jié)構(gòu)

34th搖擺位點位于拓撲結(jié)構(gòu)的末端，

堿基堆積力小，

選擇性配對的自由度大

34th搖擺位點被修飾的頻率高

導(dǎo)致配對原則的改變尤以A34

II=A/

I=

C

/

I

=U

34th幾乎無A

線粒體中

U34

=

N(any)

when

U34

U*

=A/G

only

Source:Quigley,G.J,Structural

domains

of

transfer

RNA

moleculars,Science

194:197Xo5UCmnm5UmCm5UXm5s2UK2C

(5-羥基尿苷)(5-羧甲基氨甲基尿苷)(5-甲氧基羰甲基尿苷)

(5-甲基-2硫代尿苷)

(2-賴氨酸胞苷)Com5U(5(2)-羥羧甲基尿苷)I(Inosine次黃嘌呤)m7Gm5Cm6As2Cψ(7-甲基尿苷)(5-甲基胞苷)

(6-甲基腺苷)

(2-硫代胞苷)

(假尿苷)t6A

(N6-蘇氨酸羰酸腺苷)Q(Queuosine辮苷)5.2.3.

Anti-codon及其兩側(cè)堿基修飾對密碼子

解讀的生物學意義

a)

Methylated

Nt

at

anti-codon

and

flankedb)

被修飾的Nt34的配對能力

Nt1

of

anti-codon

Nt3of

codonU

(mt,ct)CmO5U

(5(2)-羥羧甲基尿苷)Cmnm5U

(5-羧甲基氨甲基尿苷)mCm5U

(5-甲氧基羰甲基尿苷)Um

(2?-O-甲基尿苷)Xm5S2U

(5-甲基-2硫代尿苷)Q

(Queuosine)

I

(Inosine）

A,U,C,G

A,G,U

A,G

A,GA,G

AU,C

U,C,Ac)

tRNA中anti-codon堿基修飾的意義●限制對密碼識讀的隨意性，以保證遺傳的穩(wěn)定U

A/GCm5S2U

A(only)

在特定表達細胞中

S2U

A●提高搖擺能力，防止突變效應(yīng)，以保證遺傳的穩(wěn)定A

UU

A/GA

UI

A/C/U

CmO5U

A/G/U5.2.4.

tRNAabundance&codon

usage

(codon

bias)Codon

usage

Observed

for

E.coli

Ribosome

Protein1209

codons(來源：分子生物學（2007），鄭用璉，第197頁)Codon

usage

in

the

genes

of

Animals2244codons(來源：分子生物學（2007），鄭用璉，第198頁)tRNA

abundance

&

codon

usage

(codon

bias)生物GC%不等各種codon的頻率不等

進化過程中度重復(fù)基因tRNA的拷貝數(shù)與codon使用頻率的對應(yīng)

識別同一氨基酸的不同tRNA(isoacceptor)量不等

不同生物間同一isoacceptor的量不等

tRNAabundance

;

codon

usage

(codon

bias)

是進化中形成的基因表達調(diào)控機制之一

tRNA

abundance

~

正相關(guān)

~

codon

usageａ)

需要量多的蛋白質(zhì)（除mRNA轉(zhuǎn)錄速率高外）

有關(guān)aa的codonusage

高

相應(yīng)tRNA量多

需要量少的蛋白質(zhì)（除mRNA轉(zhuǎn)錄速率低外）關(guān)鍵aa的codonusage

低相應(yīng)tRNA量少ｂ）

codon

與anti-codon間的作用強度

codon

usagemodulator

）需較長時間以求結(jié)合穩(wěn)定intoAsite

of

ribosome融解溫度高需較長時間

Out

Psite

of

ribosome自然選擇codon/anti-codon間適度結(jié)合強度的codonusage

以保證最佳的蛋白質(zhì)合成速率G

C

強氫鍵配對aa-tRNAaaG

…

U

弱氫鍵配對aa-tRNAaathe

seq.of

codon

in

usage1

2

--

31

2

3in

generalUUGAA

CGG

UCC

A23

/

1209

>

10

/

120913

/

1209

>

3

/

1209Anti-codonAAG1AUG1In

prok.

Gly

(GGG)

usage

=

0

Phe

(UUC)

>

(UUU)Pro

(CCC)

usage

=

0

Tyr

(UAC)

>

(UAU)共性:

codon/anti-codon間適度結(jié)合強度個性:

G/C含量不同,tRNA豐度各異5.2.5.

two

of

three

codon-reading

in

mitochondrial

a)

線粒體中具有與通用密碼不同的編碼信息●●線粒體codon較為整齊（均為2/4/6）

2

codon;

F,

I,

Y,

H,

Q,

N,

E,

k,

D,W,

M,

C

4

codon;

V,

P,

T,A,

R,

G,

(family)

&

stop

codon

6

codon;

L,

S

(2

isoacceptorseach)In

mt

22

tRNA

only

(32

tRNA

in

universal

code)

線粒體“三中讀二”方式可減少tRNAArgstop

stopTrp

IleMet(來源：不詳)(來源：不詳)

●

Codon-readingForcodon

family;

two

of

three

readingcodon

anti-codonUCUUCAUCGUCC

UAGUC

Ser

codonN34

(U)A/U/C/G僅起將codon隔開的作用

stopTrp

AG

IleMet

ACGA

CGArgstop

CC

GG

GU

GC(來源：不詳)●

Codon-readingFor

2

codon

type;Nt34

wobble

base

G

C/UArgGAA

GAU

IleMetGUU

stop

TrpGUGGUAGCAGCU

stop

GUC(來源：不詳)●

Codon-readingFor

2

codon

type;Nt34

wobble

base*

U

G/AArgstop★UAA

IleMet★UAU★UUG★UUU★UUC

stopTrp★UCA(來源：不詳)●

Codon-reading

in

mt

(Nt34

：U/U*/G)

Forcodon

family;

two

of

three

readingUC

Ser

codon

,

N34(U)

僅起將codon隔開的作用For2

codon

type;

Nt34

wobble

base

U*

G/A

G

C/Ucodonanti-codonUCUUCAUCGUCC

UAG一種GGC編碼幾種氨基酸蛋白質(zhì)性質(zhì)不變5.2.6.

codon

in

codon

or

general

genetic

codon

(GGC

廣義密碼子)生物體除具有標準的通用密碼保證蛋白質(zhì)的準確翻譯外

同時存在GGC

轉(zhuǎn)錄的模糊性（非轉(zhuǎn)錄錯誤）

生物的適應(yīng)性a)

codon

/

anti-codon間的締合能分析●●2ed

Nt

of

codon

（N1N2N3）

對codon/anti-codon的締合能貢獻最大凡2edNt相同的codon

codon/anti-codon間的締合能相似對締合能的貢獻2Nt＞

1thNt

＞3rdNtb)

codon對氨基酸性質(zhì)的決定2edNt

of

codon

對氨基酸性質(zhì)和蛋白質(zhì)空間結(jié)構(gòu)的決定度較大NUN

非極性疏水性氨基酸α-helix

&

β-sheet的形成者位于蛋白質(zhì)分子內(nèi)部NAN

極性親水性氨基酸,位于蛋白質(zhì)分子外部N（G/C）N

編碼的氨基酸極性居中疏疏GGNCUNAUA/CGUNGCUUUU/CUGU/GAUGUGU/C

UUU/C

AUA/C

GUN

CUN

AUG

UGG

CAU/CGGN

MW

75

kd

小

GCN

UCN

CCN

GUN

CAN

UGU/C

CUNACN中UAG/C中AUA/C中

Codon

in

codon

(依

2edNt

of

codon預(yù)測氨基酸的性質(zhì))不同方法測定aa的親水性和分子量結(jié)果

(1)N1N

2N

3

(2)N1N

2N

3

F.J.R.Taylor

1989

Bio-Systems

22.p177-187N1N

2N

3UCN中GCN中GAU/C中UGGUAU/CCAA/GAAA/GAAU/CGAA/GCAU/GGGN

ACN

UCN

CCN

CGN

AAU/C

CAA/GAAU/CGAA/G

CAA/G

AAA/G

AUG

CAU/C

UUU/C親GAU/CCGNGAA/GGAU/C

親AAA/GCGN

UAU/C

UGG大

204

kd2ed

Nt

=

U

A(

hydrophobic

aa

)(

hydrophilicaa

)G/C

(

neutral

aa

)c)

Nt

of

codon

對蛋白質(zhì)功能的決定●

1th&

3rd

Nt的搖擺

對蛋白質(zhì)的結(jié)構(gòu)與功能影響不大●

2edNt不能搖擺

2edNt

of

codon對氨基酸的編碼特征

即為GGCor

codon

in

codon蛋白質(zhì)定點誘變蛋白質(zhì)改造（蛋白質(zhì)工程）d)

生物學意義

?

保證遺傳的穩(wěn)定

?

依據(jù)codonin

codon

（2edNt

of

codon）

判斷蛋白質(zhì)的性質(zhì)

?

蛋白質(zhì)性質(zhì)預(yù)測(Source:TriposAssociates/Peter

Arnold,Inc.)5.3.1.

direction

of

peptide

elongation

N?

C?R1

O

H

R2R3N—C—C—N—C—C+N—C—CHHO

OHHHO

OHHHHR3R1

O

H

R2+HHO

OHHHO

OHN—C—C

HN—C—C—N—C—C

H

H機制？P

O3Met

+

tRNAmetf

ACCA

-O2

C

O

P

OH3

H

C

N

H

H

CH

H

S

CH3

ACCA

-OH2OH

2?OH

3?

aa轉(zhuǎn)酯CH3

formylationC

OH

C

N

HH

CH

H

O

H

SCfMet—tRNA

metf&

Met--tRNA

metm

5.3.2.

Aminoacyl—tRNAaain

Prok.in

Euk.

Met—tRNA

metI

&

Met--tRNA

meteAARSAminoacyltRNAsynthetaseR非特異結(jié)合位點

DHU

loop特異結(jié)合位點

paracodan(來源：分子生物學（2007），鄭用璉，第211頁)5.3.3.

peptide

synthesisIF-1IF-2IF-39.5kd95kd-117kd

20kd加強IF-2，IF-3的酶活

促使fMet-tRNA

fmet

選擇性的結(jié)合在30S亞基上

促使30S亞基結(jié)合于mRNA起始部位(

識別tRNA

fmet

中富含GC的反密碼子臂,way

in

Psite

?!)

具有解離30S與50S亞基的活性IF2fmet

IF-2Binary

complexfmet

Complete

30s-mRNA

complexIF3

GTP

fmet

Initiation

complex

IF3

fmet(來源：不詳)

eIF2eIF2-A

eIF1

eIF3

eIF4b

eIF4a

eIF4C

eIF5

eIF4e3種亞基

65kd

15kd

＞500kd

80kd

50kd

19kd

150kd

(eIF4f

的亞基)形成三元起始復(fù)合體（eIF2,GTP,

tRNA)促使Met-tRNAmet與40S亞基結(jié)合

i

促使mRNA與40S亞基結(jié)合

促使mRNA與40S亞基結(jié)合

促使mRNA與40S亞基結(jié)合

促使與mRNA,GTP結(jié)合

促使兩亞基結(jié)合

釋放eIF2,eIF3

與5?端帽子結(jié)合(a)

:

translation

ofCapped

Sindbis

virus

mRNA(b)

:

translation

ofUncapped

picona

virus

mRNA(a)

with

eIF4e

without

eIF4e

(C)(b)(d)eIF4e

stimulates

translation

ofcapped,

but

not

uncapped,

(Source:Shatkin,Differential

stimulation

of

capped

mRNA

translation

in

vitro

by

cap-

binding

protein

Nature

285:331,

1980.including

8

activation

sites

&

occupy

20±

Nt?

Psite

(peptidyl

attachment

site)?Asite

(Aminoacyl

binding

site)?

E

site

(Exit

site

of

tRNA)?

5s

rRNAsite

(5s

rRNA+

TΨ

C

loop)?

轉(zhuǎn)位因子EF/G

binding

site?

mRNA

biding

site?

peptididyl

transferasebinding

site?

延伸因子復(fù)合體EF-Tu-aa-tRNAaa

binding

siteTranslation

domainmenbraneExit

domain

Exit5ssitesite

A

site

Peptidyl

transferase

fMet--tRNAmet

way

in

Psitesite

by

S.D

Seq.

(prok.)

EF-G

siteScanning

sequence

way

in

Psite

.

A,

site

20

Nt

(來源：分子生物學（2007），鄭用璉，第185頁)fMetMetMet-tRNAmet

iS.D.Sequence

?Scanningsequence?

CP

AfMet-tRNAmet

f

(來源：不詳)CCPAMetA

PfMet(來源：分子生物學（2007），鄭用璉，第205頁)AntibioticKilles

bacteria

and

other

cellsfMet-tRNAMet

occupiesPsite

orAsite

？(Source:Molecular

Biology(2002),Robert

F.Weaver,Page575）Chapter

5

Protein

translationtRNA

–

mini

RNA,

4s,

(70-80

Nt)

–

Nt

more

modified

by

methylation

–

tRNA

phe,

77Nt

cloverleaf

formAa

accept

arm,

DHU

loop

(contact

with

AARS),

anti-codon

loop,TΨCloop

(contact

with

5S

rRNA),

extra

loopParacodon:a

numberof

Nts,

on

tRNA,

contact

with

AARSrRNA

–

High

GC-content,

rich

methylation,

high

copy

number,synthesized

in

nucleolus

–

Pro:

23S+

5S,

16S;

Euro:

28S/5.8S+

5S,

18S

5-1mRNA

–

Pro:

Shine-Dalgarnoseq.

(S.D

seq)

GGAGG

–

Euro:5’

m7Gppp

CCACCA-3A1U2G3G4—

–

Degeneracyof

codon

–

Codon

family

–

Mechanismof

codon

degeneracy

Isoacceptor:

different

tRNA

that

load

the

same

aa,

but

recognize

different/same

codon

Wobble

hypothesis:

34th

Nt

in

tRNACodon

usuage/biasmRNA

–

Pro:

Shine-Dalgarnoseq.

(S.D

seq)

GGAGG

–

Euro:5’

m7Gppp

CCACCA-3A1U2G3G4—

–

Degeneracyof

codon

–

Mechanismof

codon

degeneracy

Isoacceptor:

different

tRNA

that

load

the

same

aa,

but

recognize

different/same

codon

Wobble

hypothesis:

34th

Nt

in

tRNACodon

usuage/bias

–

Differentcondonsare

used

at

differentfrequencyby

a

speciesPeptide

synthesis

–

Direction

of

peptide

elongation

–

Aminoacyl—tRNAaa,

Initiation

and

elongation

–

AARSthree

sites:

tRNA

site,

AA

site,

ATPDHU

loop,

nonspecific;paracodon,

specific–

Enzymes

forIF1:

separate

50S

and

30S

subunits,help

other

factorsIF2:

for

the

bindingof

fMet-tRNA

fmet

to

30SIF3:

for

the

bindingof

mRNA

to

30S5-5Peptide

synthesis

–

Enzymes

foreIF4e,

cap

bindingfactoreIF4e

stimulates

translation

of

capped,

but

not

uncappedmRNAMet-tRNAMet

occupies

ribosomal

P

site

&

InitiationtranslationTu

and

TsPuromycin

–

Resembles

an

aminoacyl-tRNA

–

Can

bind

to

the

A

site

–

Couple

with

the

peptide

in

the

P

site

–

Release

it

as

peptidyl

puromycinIf

peptidyl-tRNA

is

in

the

A

site,

puromycin

will

notbind

to

ribosome,

peptide

will

not

be

releasedTwo

sites

are

defined

on

the

ribosome:

–

Puromycin-reactivesite

(P)

–

Puromycinunreactive

site

(A)3rd

site

(E)

for

deacylated

tRNA

bind

to

E

site

as

exitsribosomeAntibioticKilles

bacteria

and

other

cellsfMet-tRNAMet

occupiesPsite

orAsite

？

?

Mixed

[35S]fMet-tRNAfMet

with

ribosomes,AUG,

and

puromycin(嘌呤酶素).

?

If

AUG

attractedfMet-tRNAMet

to

the

P

site,

then

the

labeledfMet

should

have

been

able

to

react

with

puromycin(inAsite),

releasing

labeled

fMet-puromycin.

?

If

the

fMet-tRNAMet

went

to

the

A

site,

puromycin

should

not

have

been

able

to

bind,

so

no

release

of

labeled

amino

acid

should

have

occurred.

?

fMet-tRNAMet

occupies

ribosomal

Psite

&

Initiation

translation(Source:Bretscher

and

Marcker

Nature

211:382-3,1966)

Way

in

PWay

inAunderelongation

Way

in

PSecondary

structure

near

the

5?-end

of

anmRNA

can

have

either

positive

or

negativeeffectsHairpin

just

past

an

AUG

can

force

a

pause

byribosomal

subunit

and

stimulate

translationVery

stable

stem

loop

between

cap

and

initiationsite

can

block

scanning

and

inhibit

translationGiven

the

amount

of

control

at

the

transcriptionaland

posttranscriptional

levels,

why

control

geneexpression

at

translational

level?Major

advantage

=

speed

–

New

gene

products

can

be

produced

quickly

–

Simply

turn

on

translation

of

preexisting

mRNA

Valuable

in

eukaryotes

Transcripts

are

relatively

long

Take

correspondingly

long

time

to

make

–

Most

control

of

translation

happens

at

the

initiation

stepMost

bacterial

gene

expression

is

controlled

attranscription

levelMajority

of

bacterial

mRNA

has

a

very

shortlifetime

–

Only

1

to

3

minutes

–

Allows

bacteria

to

respond

quickly

to

changing

circumstancesDifferent

cistrons

on

a

polycistronic

transcriptcan

be

translated

better

than

othersmRNA

secondary

structure

can

governtranslation

initiation

–

Replicasegene

of

the

MS2

class

of

phagesInitiationcodon

is

buried

in

secondary

structure

untilribosomes

translating

the

coat

gene

open

up

the

structure–

Heat

shock

sigma

factor,

s32

of

E.

coli

Repressed

by

secondary

structurethat

is

relaxed

by

heating

Heat

can

cause

an

immediate

unmaskingof

initiationcodons

and

burst

of

synthesisShift

from

σ70

to

σ32

attemperature

higherthan

37°C當突變使莖I的堿基配對增強時，高溫誘導(dǎo)作用減弱：如+5的C變?yōu)锳，誘導(dǎo)作用由3.5倍降低為1.4倍當突變使莖I的堿基配對減弱時，高溫誘導(dǎo)作用增強Small

RNAs

with

proteins

can

affect

mRNAsecondary

structure

to

control

translation

initiationFerritin

mRNA

translation

is

subject

to

induction

byironInduction

seems

to

work

as

follows:

–

Repressor

protein

(aconitase

apoprotein)

binds

to

stem

loop

iron

response

element

(IRE)

–

Bindingoccurs

near

5’-end

of

the

5’-UTR

of

the

ferritin

mRNA

–

Iron

removes

this

repressor

and

allows

mRNA

translation

to

proceedOne

factor

is

T,

transfer

–

It

transfersaminoacyl-tRNAs

to

the

ribosome

–

Actually

2

differentproteinsTu,

u

stands

for

unstableTs,s

standsfor

stableSecond

factor

is

G,

GTPase

activityFactors

EF-Tu

and

EF-Ts

are

involved

in

the

firstelongation

stepFactor

EF-g

participatesin

the

third

stepElongation

takes

place

in

three

steps:1.

EF-Tu

with

GTP

binds

aminoacyl-tRNA

tothe

ribosomal

A

site2.

Peptidyl

transferase

forms

a

peptide

bond

between

peptide

in

P

site

and

newly

arrived

aminoacyl-tRNA

in

the

A

siteLengthens

peptide

by

one

amino

acid

andshifts

it

to

the

A

site3.

EF-G

with

GTP

translocates

the

growing

peptidyl-tRNA

with

its

mRNA

codon

to

the

P

siteEF-T

dependent

binding

ofcharged

tRNA

to

ribosomerequired

GTPPolymerization

required

both

EF-T

and

EF-G

and

a

highconcentration

of

GTP

18-104(a)

EF-Tu與GTP結(jié)合形成二元復(fù)合物(b)

進一步與aminoacyl-tRNA

形成三元復(fù)合物(c)

三元復(fù)合物與P位點已有peptidyl-tRNA的核糖體結(jié)合(d)

GTP被水解，形成

EF-Tu–GDP復(fù)合體，從核糖體上解離，在A位點留下新的aminoacyl-tRNA(e)

EF-Ts

exchanges

GTP

for

GDP

on

EF-Tu，生成新的

EF-Tu–GTP復(fù)合體EF-Ts在以EF-Tu–GDP

為底物時能夠促進aminoacyl-tRNA

、Tu、GTP三元復(fù)合物的形成

(panel

a).EF-Tu–GTP

(panel

b)

or

EF-Tu+GTP

(panel

c)

能夠不依賴于EF-Ts自發(fā)形成三元復(fù)合物.PAMetAUGAPAUG

UUU

CUGUAGTu

PheGDP

Tu

TsMet

PhePAAUG

UUU

CUGUAG

GTP

TuTs

+ADP

ATPEF-G

be

needed

for

translocationGTPGDP

TuTs

Temperate

S

(來源：分子生物學（2007），鄭用璉，第206頁)Releasefactor1/2

(or

transpeptidaseor

RF

or

rRNAribozyme

?)peptide

+

tRNA

+

mRNA

+

large

&

small

subunit…Complex

disassembleHydrolysis

PM

F

ALAUG

UUU

CUGUAG

PM

F

LAUUU

CUGUAGRF

PM

F

LAUUU

CUGUAGM

F

LRFRF1-

UAA

/

UAGRF2-

UAA

/

UGA(來源：不詳)5.5.保證肽鏈準確翻譯的機制(來源：不詳)ξ=10-4(來源：不詳)

DNAreplication

ξ=

10-11

RNAtranscription

ξ=

10-4Peptide

translation

P(準確率)

=

（1—ξ）n(氨基酸的數(shù)目)

N1002001000P(ξ=10-2)

36%

4.9%

0.004%P(ξ=10-3)

91.5%

84%

36%P(ξ=10-4)

99%

97%

90%MACHENISM

?√AARSAminoacyltRNAsynthetaseR非特異結(jié)合位點

DHU

loop特異結(jié)合位點

paracodan(來源：分子生物學（2007），鄭用璉，第211頁)

5.5.1.

氨基酸與tRNA間的負載專一性a)

氨基酰tRNA合成酶（AARS）對氨基酸的特異識別與結(jié)合

AARS;

aa

binding

site,

tRNA

binding

site,ATPsite

aa

binding

site

對結(jié)構(gòu)相似的氨基酸的雙篩作用例；CysHS—CH2—CH—COOHNH2AlaH—CH2—CH—COOH

NH2Ala-RS結(jié)構(gòu)相似錯誤識別錯誤負載錯誤翻譯IleHCOOHValCH3—C—CH

CH3

NH2Ile-RS

HIn

vitro

Ile

&

Val

濃度相等的情況下

CH3—CH2—C—CH

CH3

NH2200X∨1X

Val-tRNAIle

錯誤負載機率

1/200!COOHIn

vivoVal

:

Ile

=

5:1但實際測定的錯譯機率僅為1/3000

?!Val-tRNAIle

錯誤負載機率1/40

!!How

?aa?+ATPAARSMis-activationaa?-AMP+

tRNAaaMis-loading

AARSDouble

Sieve

effect

aa?-tRNAaa

Hydrolyzedor

Edited

aa’+AMPaa

binding

site具有

結(jié)合位點（

Biding

Site

orActivation

Site

)

水解位點（HydrolyticSite

or

Editing

Site

）Ile

/

Val

進入B

位點

Kinetic

ComformationalChemical發(fā)生誘導(dǎo)契合proofreadingKinetic發(fā)生誘導(dǎo)契合Ile

分子構(gòu)型大于Val?

Ile進入B位點但不能進入H位點?

Val進入B位點并進入H位點而被降解H

位點柔性部位小Ile

/

Val

進入B

位點ComformationalChemicalproofreadingDouble

SieveBH(來源：分子生物學（2007），鄭用璉，第212頁)

(副密碼子)的概念；

tRNA中決定負載特定氨基酸的空間密碼tRNA中的特定序列與AARS

的tRNAbinding

site

的特異基團間的分子契合

AARStRNAbinding

siteaa

binding

siteParacodonof

tRNAloadingAmino

Acid（R）●的特征

是為AARS

特定氨基酸所識別的若干Nts（并非均為一對Nts,

也并非僅只有一處的Nts）AARS

對的識別與結(jié)合是通過氨基酸與堿基之間的連接實現(xiàn)的。屬于生物II

型空間密碼

為同一種AARS

所識別的一組同功受體具有相同的

副密碼子(除AARSala外，其他證據(jù)不足！！)

tRNAAla(GGC)tRNAAla(UGC)具有G3：U70

也是進化進程留下的tRNA可能起源于可以攜帶氨基酸的

由AARS

特異識別tRNA

中的特定序列

使氨基酸的負載更為準確

成為進化的優(yōu)勢

位于tRNA的各種環(huán)或臂上不同tRNA的的定位不同aa

arm73th

siteA.C.D.E.F.G.H.I.K.L.M.N.P.Q.R.S.V.W.Y.A,D,G,H,N,S,T,V,W

DHU

loopExtra

loopTC

loopA,

F,

L,

R

Anti-codon

loop

C,D,E,F,G,H,I,K,M,N,P

Q,R,T,V,W,Y

(來源：分子生物學（2007），鄭用璉，第212頁)第五章蛋白

質(zhì)

翻

譯5.5.

Central

Dogma

的發(fā)展

來源：不詳RNAproteinCrickF.

H.

C

On

proteinsynthesisSymp.Soc.Exptl.Biol.1958(12)

:

138-163

Genetic

Central

Dogma

DNA中心法則的要點；?

所謂遺傳信息，是指核酸中的堿基序列以及蛋白質(zhì)中的氨基酸序列。生物的全部遺傳信息均包含于這種大分子的遺傳序列的信息中。?

從DNA到RNA到蛋白質(zhì)的遺傳信息流是嚴格的單程路線。信息一旦進入蛋白質(zhì)，就不可能再行輸出。蛋白質(zhì)是一切性狀形成的工作分子。?

序列假說是中心法則的核心，中心法則是序列轉(zhuǎn)換的原則中心法則體現(xiàn)的基本原則；遺傳信息的唯一性遺傳物質(zhì)的自決性信息表達的單程性序列轉(zhuǎn)換的共線性對分子生物學多年來的最大的一個浪頭更加感到困惑中心法則的發(fā)展與修正科學王國不信奉教義與信條（dogma）Anti

Central

Dogma

(中心法則的發(fā)展)a.蛋白質(zhì)的遺傳信息

并不一定來自核酸

!?L-PhegeneralD-Phespecial

HE(聚合酶)

通過轉(zhuǎn)硫醇和轉(zhuǎn)肽

反應(yīng)，將肽鏈按D-Phe

：Pro

：Val

：Ornithine(鳥氨酸)

：Leu

按1

：1

：1

：1：1