分生-課件分子生物學14-transcription

Review:

transcription

in

prokaryotesCore

EnzymeRNA

Polymerase

HoloenzymeTop-IsomeraseIGyraseTop-IsomeraseIGyraseRibosomeRibosomeExo-

nucleaseRibosomeNusAExo-nucleaseMolecular

BiologyTranscription

in

EukaryoteMain

contents

Eukaryotic

RNA

polymerases

RNA

Pol.

I

genes：The

ribosomal

repeat

RNA

Pol.

II

genes：hnRNA,

snRNA

RNA

Pol.

III

genes：5S,

tRNA,

snRNA,plasma

RNA

Promoters

and

enhancers

General

transcription

factors

and

RNA

Pol.

IIinitiationMultiple

Forms

ofEukaryotic

RNA

Polymerases多種真核RNA聚合酶Three

major

RNA

polymerases

operating

ineukaryotic

nuclei,

having

different

structures,transcribing

different

classes

of

genes.

RNA

Pol.

I,

nucleolus,

large

rRNA

precursor

(45S

-28S,

18S,

and

5.8S)

RNA

Pol.

II,

nucleoplasm,

hnRNA

(mRNAprecursor)

,

snRNA,

miRNA

etc.

RNA

Pol.

III,

nucleoplasm,

tRNA,

5S

rRNA

andsome

other

small

RNA.Separation

of

RNA

Polymerases

by

ion-exchange

chromatography用離子交換色譜法分離RNA

聚合酶DEAE-Sephadex

葡聚糖凝膠二乙氨乙基Separation

of

RNA

PolymerasesRNA聚合酶的分離Peeks

of

activitiesofRNA

pols

from

mousenucleoplasm鼠核質(zhì)中RNA聚合酶的活性峰Peeks

of

activitiesofRNA

pols

from

mousenucleoli鼠核仁中的RNA聚合酶的活性峰α-amanitinα-鵝膏覃堿Amanita

phalloides鬼筆鵝高菌α-amanitin

and

the

activities

of

RNApolymerases（α-鵝膏覃堿與RNA聚合酶的活性）Roles

of

RNA

Polymerases（RNA聚合酶的作用）RNA

Polymerase

Subunits

(1)Human

and

yeast

RNA

polymerase

II

subunitsRNA

Polymerase

subunits

(2)Subunits

are

placed

in

3

groups:

Core

subunits

（

亞基）–related

in

structure

andfunction

to

bacterial

core

subunits.Rpb

1,

2, 3,

11

–

Eukaryotic

RNA

polsβ’,

β,α,α

--

Prokaryotic

RNApol

Common

subunits

（共有亞基）–found

in

all

3nuclear

RNA

pols.Rpb5，Rpb6，Rpb8，Rpb10，Rpb12

Nonessential

subunits

（非必需亞基）–conditionallydispensable

for

enzymatic

activity，Rpb4，Rpb7Eukaryotic

RNA

polymerase

activities真核RNA聚合酶的催化活性

Direction（方向）:5’-3’

Template（模板）:antisense

DNA

chain

Substrate（底物）:ATP,GTP,CTP

andUTP

Additional

initiation

proteins

are

required

forbinding

to

promoters

and

initiating

transcription.（與啟動子的結(jié)合及起始轉(zhuǎn)錄需要額外的蛋白參與）Comparison

of

RNA

polymerases

betweeneukaryote

and

prokaryote真核與原核RNA聚合酶比較Prokaryotic

原核的

Eukaryotic

真核的OneNo

primersMultiple

subunitsAdditional

initiationproteins

are

not

requiredthreeNo

primersMultiple

subunitsAdditional

initiationproteins

arerequiredCTD

of

RNA

pol

II（RNA聚合酶II的羧基末端結(jié)構(gòu)域）CTD

：

the

carboxyl-terminalwith

repeated

amino

acid

sequence

ofTyr-Ser-Pro-Thr-Ser-Pro-SerPhosphorylation at

Ser

,

Thr

and

Tyr

in

the

CTDRNA聚合酶II的羧基末端結(jié)構(gòu)域含有重復的Tyr-Ser-Pro-Thr-Ser-Pro-Ser氨基酸序列，通過該序列中的Ser,Tyr和Thr的磷酸化與否影響轉(zhuǎn)錄的啟動子的結(jié)合、延長等等CTD

of

RNA

pol

II（RNA聚合酶II的羧基末端結(jié)構(gòu)域）Phosphorylation at

Ser

,

Thr

and

Tyr

in

the

CTDEnzyme

with

IIa

bindsto

the

promoterEnzyme

with

IIo

is

involved

in

transcripongationThree

D-

structure

of yeast

RNA

polymerase

IIRNA

polymerase

I

：rRNA

Ribosomal

genes

are

different

from

other

nucleargenes

Different

base

composition

from

other

nuclear

genes

Unusually

repetitive,

40×5

Found

in

the

nucleolusrRNA

transcription

unit

in

eukaryotesNon-transcribed

spacernTranscribed

region18S5.8S28S45S

(13000

nt)5000

nt2000

nt160

ntrRNA

and

nucleolusPromoter

of RNA

polymerase

IUCE-

upstream

control

element

，

enhancing

thetranscription

efficiency

by

10—100

times啟動子:轉(zhuǎn)錄的起始位點，轉(zhuǎn)錄所必需的，但是其單獨作用時轉(zhuǎn)錄效率較低上游調(diào)控元件或者上游激活：它在啟動子的上游,可以結(jié)合不同的蛋白，提高轉(zhuǎn)錄的效率。-100Pre-rRNA

gene-31+1650-80

bp上游調(diào)控元件UCE啟動子Core

elementUpstream

Binding

Factor

(UBF)上游結(jié)合因子+1UCECore

elementUBFUBFCore

elementUBFUCEOne

UBF

can

bind

to

UCE

and

the

other

binds

to

a

upstreampart

of

the

core

element.

The

two

UBF

may

then

bind

to

eachother.UBFs

stimulatetranscriptiongreatly

.一個UBF結(jié)合到UCE，另一個結(jié)合到 元件的上游，兩個UBF再互相結(jié)合Selectivity

factor

1

(SL1)選擇因子TATA

binding

proteinTBPTBP

is

required

for

initiationfor

all

three

eukaryotic

RNAPols

and

seems

to

be

a

criticalfactor

ineukaryotictranscription.TBP是三種真核RNA聚合酶起始轉(zhuǎn)錄是必需的，在真核轉(zhuǎn)錄中起關鍵作用TBP-associated

factors(TAFs)TBP協(xié)同因子TAF1TAF1TAF1TBP:TATA結(jié)合蛋白Selectivity

factor

1

(SL1)UBFCore

elementUBFUCEUBFUCETBPUBFTBPTAF1

SL1

binds

to

andstabilizes

the

UBF-DNA

complex

，interacts

with

thefree

part

of

coreelement.選擇因子與UBF-DNA復合物結(jié)合，與

元件的游離部分相互作用SL1UBFUBFUCETBPRNA

Pol

IRNAPol

IUBFUBFUCETBPSL1

binding

allowsRNA

Pol

I

to

bindto

the

complex

andinitiate

rRNAtranscription.SL1

的結(jié)合使得

RNA聚合酶I結(jié)合到這個復合物上并起始RNA的轉(zhuǎn)錄。Other

rRNA

genes

TIF-1

（transcriptional

intermediary

factor

1）

fromAcanthamoeba

（棘阿米巴）is

a

homologous

of

SL1，binds

to

promoter

in

region

from -12

to -72bpnecessary

for

initiation

of

transcription.TIF-1（轉(zhuǎn)錄中介因子）是SL1的同源蛋白，在-12和-72bp之間與啟動子結(jié)合，是轉(zhuǎn)錄的起始所必須的

Additional

UBF,

responsible

for

sequence-specific-ing

of

SL1

to

the

promoter.另一個UBF負責SL1與啟動子的序列特異性定位RNA

Pol

III:

5S

rRNA

and

tRNARNA

Pol

III

contains

at

least

16

subunits,

which

islocated

in

nucleoplasm.

It

is

responsible

forsynthesis

of

precursor

of

5S

rRNA,

tRNA,

snRNA,and

cytosolic

RNAs.RNA聚合酶III含有至少16個亞基，定位于細胞核質(zhì)。負責5S

rRNA,

tRNA,

snRNA,

and

cytosolic

RNAs.的前體的

。tRNA

gene

promoter5’- NAGTGG-3’5’

–

GGTTCGANNCC-3’5sRNAt

RNAU6RNAbox

Abox

BEncode

Dring

in

tRNAEncode

TψC

ringTranscription

of

tRNABox

A Box

BTF

IIICTF

IIICTF

IIIBTBPTFIIICTF

IIIBPolIIITBPTF

IIIBTBPPol

IIITF

IIICTFIIICTFIIIBPol

IIITans.TF

IIIC

binds

to

both

Aand

B

boxes,

anddetermine

the

position

forinitiation

factor

TF

IIIB.需兩轉(zhuǎn)錄因子，TFIIIC先結(jié)合到A

、B

框，決定TFIIIB（含TBP）的位置，然后TFIIIB結(jié)合到轉(zhuǎn)錄起始位點，隨后Pol.III

與TFIIIB結(jié)合并起始轉(zhuǎn)錄，TFIIIC。Transcription

of

5S

RNABox

ABox

CTFIIIATF

IIICTFIIIATF

IIICTFIIIATFIIIBTF

IIICTFIIIARNA

pol

IIITFIIIBTF

IIIATF

IIICTFIIIBRNA

pol.transcription81-99bpTF

IIIA

acts

as

aassembly

factorwhich

allowsTFIIIC

to

interactwith

the

5S

rRNApromoter.Box

A

may

alsostabilize

theTFIIIC

binding.Once

TFIIIC

hasbound,

TFIIIB

caninteract

with

thecomplex

andrecruit

RNA

Pol

IIIto

initiatetranscription

.RNA

pol

II--

To

catalyzes

the

synthesis

of

the

mRNAprecursors

for

all

protein-coding

genes

and

somesnRNA

gene.—RNA聚合酶II催化mRNA的前體，和一些核內(nèi)小

RNA

的Enhancer/UREsTATA

boxgene-25+1transcription5’-

TATA(A/T)A(A/T)-3’TATA

box

determines

the

site

of

RNA

pol

II

or

proper

initiationTATA框決定RNA聚合酶II或正確起始的位置50%of

transcription

started

with

A,

G。UREs

:

enhance

the

transcription,

e.g.SP1

box,CCAAT

box在TATA

box上游（between-100

and-200），它們能增強轉(zhuǎn)錄Promoter

and

Enhancer（啟動子和增強子）Examples

of

sequences

with

TATAboxTranscription

start

sitePositive

regulation：gene

enhancerEnhancers

exert

strong

activation

of

transcription

of

a

linked

gene

from

thecorrect

start

site；

activate

transcription

when

placed

in

either

orientationwith

respect

to

linked

genes；be

able

to

function

over

long

distances

ofmore

than

1

kb

whether

from

an

upstream

or

downstream

of

the

closest

oftwo

tandem

promoters.enhancerenhancerPromoterenhancerPromotergenePromoter

genegeneDNADNADNARNA

pol.

IIGeneral

transcription

factorsThey

combine

with

RNA

polymerase

to

form

a

pre-initiation

complex

，which

is

able

to

initiatetranscription

when

nucleotides

are

available。Class

I

TranscriptionfactorsA

core-binding

factor,

SL1

or

TIF-IBA

UPE-bindingfactor,upstream-bindingfactor

(UBF)

or

upstream

activating

factor(UAF)SL1RNAPol

IUBFUBFUCETBPClass

III

transcription

FactorsTFIIIA

isthemember

ofthe

family

of

DNA-bindingproteins

that

feature

a

zincfeature

to

be

described

Zinc

finger

is

roughlyfinger-shaped

protein

Contains

4

amino

acids

thatbind

a

zincionTFIIIB

and

TFIIIC

arerequired

for

transcriptionof

the

classical

polymeraseIII

genesClass

II

general

transcriptionfactors

Preinitiation

complex

contains:

RNA

PolymeraseII

6

general

transcription

factors:

TFIIA

TFIIB

TFIID

TFIIE

TFIIH

TFIIJ

The

transcription

factors

(TF)

and

polymerase

bindthe

preinitiation

complex

ina

specific

orderFour

Distinct

Preinitiation

Complexes

１.

DA

complex

２.

DAB

complex

３.

DABPolF

complex

４.DABPolFEHJ

In vitro

theparticipation

of

TFIIAseems

to

be

optionalTFIID在TFIIA的幫助下，結(jié)合到TATAbox形成DA復合物，TFIIB再結(jié)合進去形成DAB復合物，在TFIIF的幫助下，RNA

pol

II

結(jié)合到-34--+17的位置形成DABPolF

復合物，最后，TFIIE

、TFIIH

（和TFIIJ

）結(jié)合上去形成

DABPolFEHJ復合物。Structure

and

Function

of

TFIIDTFIID

contains：(1)

TBP:Highly

evolutionarily

conservedBinds

to

the

minor

groove

of

theTATA

boxSaddle-shaped

TBP

lines

up

withDNAUnderside

of

the

saddle

forcesopen

the

minorgrooveThe

TATA

box

is

bent

into

acurve

(80°)(2)

TAFIIs

:

TBP-associated

factorsspecific

for

class

II8-10

copiesTBP

and

DNATBP

is

a

universaltranscription

factorrequired

by

all

threeclasses

of

RNApolymerasesStructure

and

Function

of

TFIIBTFIIB

binds

to

TBP

at

theTATA

box

viaits

C-terminal

Polymerase

IIvia

its

N-terminalThe

pr