分析分子-理論課reverse transcription p

In

1957,

Crick

put

forward

the

centralDogmaA

significant

experimentScientists

used

T2

phage

to

infectE.coli,

then

they

found

E.coli

stopmaking

RNA

but

there

are

more

T2phage

RNA

produced.

The

new

RNA’sbase

pair

ratio

is

similar

with

the

T2phage’s

DNA

base

pair

ratio.

And

theyused

DNA

hybridization

to

prove

DNAis

the

template

of

RNA.The

final

determinationIn

1961,

Brener.S,

Jacob.F

and

Meselson

findthe

transcription

by

the

following

experiment:

they

use

C13

and

N 5medium

to

fostthe

E.coli,so

the

E.coli’s

ribosome,

DNA

andRNA

are

heavy.Then

they

used

C12,N14

butP32,S35medium

and

let

T2

phage

to

infect

theE.coli.

After

few

days,

they

broke

E.coli

andcentrifugal

them.They

found

the

heavy

ribosomes

areradioactive

but

the

light

ribosomes

are

not.That

is

to

say,T2

phages

didn’t

make

ribosomes,they

just

used

the

ribosomes

from

the

E.coli,

inother

words

,

the

ribosomes

are

not

specificity.However,

RNA

is

specificity

because

it

has

thespecific

bases

sequence.

So

they

concluded

thatthe

messenger

between

DNA

and

protein

is

RNA,and

they

called

the

messenger

RNA

which

is

justthe

mRNAHowscientistsfind

thereversetranscriptionHe

got

the

acellular

filtrationof griding

affectedtissue,(碾碎的雞肉瘤組織的無細胞濾液）

and

injectedintothenormal

chicken.The

Nobel

Prize

forphysiology or

medicineRenatoDulbeccoAfter

enteringthe

cell,

the's

DNAwillintegretewiththe

host's

DNA.Then

the

cell

isinduced

tohavecanceration

.pro

（前

）hypothesisI

find

thesecret

ofRNAAfter

entering

the

cell,will

synthesis

DNA

accordingto

its

RNA

template,this

time

is

called

pro

.Then

this

DNA

can

integretewith

tne

host's

DNA.Reversetranscriptase--ARNA-dependentDNApolymeraseFrancis

Harry

Compton

CrickHe

amended

thecentral

dogmareverse

transcriptiontheprocess of

the

discovery

of

reverse

transcriptionphenomenoncan

causecancerprocarcinogenicmechanism

of

DNAreversetranscriptaserWhat

is

it?How

is

itdiscovered?How

does

itwork?What’s

the

biological

value?The

Big

DiscoveryesTime:1970Discoverer:

Howard

Temin

and

DavidBaltimoreHow:

discovered

in

the

RNA

tumorResult:

1975

Nobel

Prize

for

Physiology or

Medicinealong

with

Renato

DulbeccoDefinitionReverse

transcriptase

(RT)

is

an

enzyme

usedto

generate

complementary

DNA

(cDNA)

from

an

RNA

template.reversetranscription!RNA→DNAIn

reverse-transcribingesReverse-transcribingRNAEg.RetroesesReverse-transcribingDNA

esEg.Hepadna

esRetroviral

RT

has

three

sequential

biochemicalactivities

:RNA-dependent

DNA

polymeraseribonuclease

H

(RNase

H)DNA-dependent

DNA

polymeraseThe

same

sequence

of

reactions

is

widely

used

in

thelaboratory

to

convert

RNA

to

DNA

for

use

in

molecularcloning,

RNA

sequencing,

polymerase

chain

reaction

(PRC)

,enome

ysis.Well

studied

reverse

transcriptases

include:1.HIV-1

reverse

transcriptase

from

human

immunodeficiencytype12.M-MLV

reverse

transcriptase

from

the

Moloney

murine

leukemia(莫

氏鼠白血病

)3.AMV

reverse

transcriptase

from

the

avian

myeloblastosis

(禽骨髓omeres(端粒)母細胞瘤

)4. omerase

reverse

transcriptase

that

maintains

theof

eukaryotic

chromosomesCrystallographic

structure

of

HIVreverse

transcriptaseHIVesRT

inhibitors

are

widely

used

as

antiretroviraldrugsRT

activity

is

also

associated

with

the

replicationof

chromosome

ends

( omerase)

and

somemobile

genetic

elements

(retrotransposons)It

plays

an

essential

role

in

the

reproduction

ofthese

molecular

parasites,

and

has

manybiological

values

as

well.Great

ValuesRT

is

needed

for

the

replication

of

retro(e.g.HIV)So

what

is

the

process

of

the

ReverseTranscription?逆轉(zhuǎn)錄酶的作用是以dNTP為底物，以RNA為模板，tRNA(主要是色氨酸t(yī)RNA)為引物，在tRNA3‘-末端上，按5’→3‘方向，

一條與RNA模板互補的cDNA單鏈，它與RNA模板形成RNA-cDNA雜交體。隨后又在反轉(zhuǎn)錄酶的作用下，水解掉RNA鏈，再以cDNA為模板第二條DNA鏈。至此，完成由RNA指導的DNA

過程。In

short,

reversetranscriptionis

the

processwhere

DNA

is

synthesized

from

an

RNA

template.But

there

is

muore

story

to

be

discovered…The step

of

reversetranscription:，it’s

the

initiation

ofthe

anti-strand

DNA

synthesis.DNTP

isusedas

asubstrate,

RNA

is

used

as

a

template,tRNA

(tryptophan

tRNAs)

is

usedasa

primer.The

synthesisofathecDNAis

onthe5’to3’direction

ofsingle-stranded

RNAtemplate

.It

is

very

amazing

toknow

theRNase

H

only

catalyzes

the

cleavageofRNA

,

allowingcompletion

of

thenewly

synthesized

DNA.Thesecond

stepof

reversetranscription:The

secondstep

is

template

exchange.

There

aretotally

two

template

exchanges

in

theRT,

while

it

isthe

one.Ppt,

alsocalled

polypurinetract,

resistsdigestion

byRTand

primers

plus

strand

DNAsynthesis.The

thirdstep

ofreverse

transcription:When

thesynthesisofthe

plusstrand

DNA

is plished,thereare

twopbs

sequences,onefromthe

RNAgenome,

the

other

fromthetRNA

primer.Then,

the

RNase

H

endonuclease

activity

ofRT

removes

both

primer

RNAs,the

tRNA

and

the

ppt.Because

there

are

two

PBS

sequences,

DNA

ends

are

juxtaposed

by

annealing

atcomplementaryPBSsequences.The

last

stepof

reversetranscription:Reverse

transcripataseandHIVGFP:綠色熒光蛋白RNAIntegrase整合酶proteaseLipoprotein

envelope脂蛋白包膜逆轉(zhuǎn)錄