Part 3

Gene expression (Ⅰ)transcription

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Summary for Transcription

the Recognization of template ↓

Initiation↓

Pass the promoter↓

Elongation↓

Termination

Ⅰ. Transcription in Prokaryote

Ⅱ. Transcription in Eucaryote

Ⅲ. Products of transcription

Ⅰ. transcription in ProkaryoteIntroduction:• RNA polymerase• Promoter • Initiation and

termination of transcription

RNA polymerase

核心酶

σ 因子

Function of subunit:

α : combine to the promoter, unwind the double-strand

β : for the nucleotide’s combining in initiation and elongation process

β’ : recognize and combine to the template ,unspecifically

σ : recognize and combine to the template specifically

ω : unkown

Promoter

• -10 signal (TATA box,Pribnow box)

• -35 signal

• transcription start site

TATAATTGACA

-35 box

+1

-10 box

-40 ~-30

转录起点

上游

upstream下游

downstream

原核生物的启动子

17+1bp

-10 ~ -7

Initiation

The process of transcription initiation, with every subunit of the RNA polymerase making function orderly ,is from the template being recognized and bound to the first nucleotide residue being transcribed on the start site.

+1

+1

+1

+1

-10

-10

-10

-10

-35

-35

-35

-35

β’

σ

α

β

脱落的 σ ATP or GTP

1

2

3

4

Termination

Two different pattern: ρ- independent termination

ρ –dependent termination

1.reverse repeat with GC rich

2.poly (A) following the GC rich se

quence directly in template

ρ factor:

(1) moves along the nascent RNA towards the transcription complex

(2) Hydrolyzes the ATP

(3) Helicase

不依赖ρ因子的终止子

依赖ρ因子的终止子

Ⅱ. Transcription in Eucaryote

Introduction:• RNA polymerase• Promoter and transcription initiation• Termination of transcription• Post-transcriptional Processing

RNA polymerase

Multi-subunit enzyme

Contain: two large subunits whose MW are more than 1*105 several small subunits which may be the same in 2 o

r 3 kinds of RNA polymerase

different RNA polymerase is with responsibility for different gene

Polymerase

location Respondent gene

RNA polymerase Ⅰ

Nucleoli ( 核仁 )

rRNA gene cluster (except 5s rRNA gene)

RNA polymerase Ⅱ

nucleoplasm （核质）

all genes encoding protein, some encoding U-RNA

RNA polymerase Ⅲ

nucleoplasm （核质）

5s rRNA gene, tRNA gene

RNA polymerase and its respondent gene

rRNA gene cluster (Pol gene )Ⅰ

18s-5.8s-28s

18s-5.8s-28s

18s-5.8s-28s

18s-5.8s-28s

promoterPromoterrepeat

Promoter repeat

60/81bp repeatNTS

Typical mRNA gene (pol gene)Ⅱ

Coding region5’- 侧

翼 3’ - 侧翼

5’UTR

3’UTR

EXON EXON EXON EXON

Pol geneⅢ

+1

内启动子

A box

+50~+65

C box

+81~+99

5s rRNA gene

tRNA gene

+1

内启动子

A box B box

D-loop TψC-loop

Promoter & transcriptional initiation

• Promoter : A sequence region which promote the initiation of transcription

• Transcriptional initiation

RNA pol promoterⅠ

+1

+6-31-100

UCE:50~80bp Core promoter Pre-rRNA gene

Enhance transcription

Essential for transcription

Initiation of pol gene’s transcriptionⅠUBFUBF

SL1POL Ⅰ

NOTE: UBF----upstream binding factor

SL1----selectivity factor

RNA Pol PromoterⅡ

Consist of:1) Transcription start-site: A or G

2) TATA box: determine the exact starting

3) UAS (UPE): determine the frequency of initiation

4) Enhancer : enhance the frequency of initiation

ATATAACCAATGCCACACCC

或

GGGCGGG

+1

转录起点TATA boxCAAT boxGC box增强子

UPE or UAS

Py A Py

promoter model

-35 ~ -25-80 ~ -70-110 ~ -80

Enhancer in SV40

72bp ，分 A 、 B 、 C三区

A C B

-200-220-232-250 -248-271

两个核心 A

或 核心 A+ 部分A

核心 C+TCⅡ

Sph +SphⅡ Ⅰ

三个主区相互作用发挥增强转录的作用

The enhancer’s architecture in SV40

Transcription initiation of pol geneⅡ

Transcription factors recognize and bind to the promoter to assemble an initiation complex(basal transcriptional initiation complex or activated transcriptional initiation complex)

Basal transcriptional initiation complex

Activated transcriptional initiation complex

TATA

ⅡAⅡB

ⅡF

ⅡE

ⅡH

TBPTAFs

PolⅡCBPUSA

Mediator

AAAAAATAAA

3’

加尾信号 Poly(A)

真核生物中与转录终止有关的序列

Termination

终止

0.5~2 kb15~30 bp

Termination of transcription

In eucaryote, the transcription termination is different from that in prokaryote.

polyadenylation signal & Poly(A) addition site: the terminus signal but not the terminator

Terminate downstream

TF CⅢ

Pol promoter & transcriptioⅢn initiation

+1内启动子

A box

+50~+65

C box

+81~+99

5s rRNA gene

TF BⅢ

Pol Ⅲ

TF AⅢ

内启动子

A box B box

tRNA gene

D-loop TψC-loop

+1

TF CⅢTF BⅢ

Pol Ⅲ

Post-transcriptional Processing

• tRNA processing

• rRNA processing

• mRNA processing

rRNA processing

ITS1 ITS2ETS1 ETS2

RNase

RNase

RNase

tRNA processing

mRNA processing

• 5’ capping• 3’ polyadenylation• splicing ： primary product: hnRNA ( intron, exon)

• RNA editing and modification

mRNA

POSTTRANSCRIPTIONAL PROCESS

5’ capping

m7G 5’-p-p-p-5’AN

m7G ppp p-p-p-5’ A N

腺苷酸转移酶甲基转移酶

Cap 0

Cap 1

Cap 2

5’ ， 5’ – 磷酸二酯键

m7G 5’-p-p-p-5’Am2’N

m7G 5’-p-p-p-5’Am2’Nm2’

GTP 尿苷酸 -7- 甲基转移酶

3’ polyadenylation

AAUAAA CASTOP15~30 bases

0.5~2kb

polyadenylation

Cleavage site

Poly(A)addition site

Pre-mRNA

splicing Interrupted gene: exon & intron Classes of intron:

» 主要的内含子 : 5 ’GU……AG 3’» 次要的内含子 : 5’ AU……AC 3’» Ⅰ 类内含子» Ⅱ 类内含子» Ⅲ 类内含子» 双内含子» tRNA 前体中的内含子

Introns are spliced in posttranscription processing ：见课本 P92 、 94 、 95

Alternative splicing: Reference: “regulation of eucaryote gene expression”

主要的内含子

exon exon

AGGUAAGU PyPyPyPyPyPyNCAG

5’ 剪接位点

3’剪接位点

多聚嘧啶系列

CURAY

分支点序列

RNA modification

Modification:specific chemical modification. 甲基化 去氨基 硫代 积极的同分异构化 二价键的饱和化 核苷酸的替代

Methylation guided by snoRNA

RNA editing

Reference: “regulation of eucaryote gene expression”

Editing: to alter the coding information by change the mRNA sequence.

Products of transcription

• In prokaryote short half-life:Multi-cistronStarting codon & SD sequence• In eucaryote 5’ cap3’ tailPrimary products must be peocessed to pro

duce mature mRNA

SD sequence

AUG~10bases

UAAGGAGGU:complementary with 16s rRNA

5’

The site for ribosome binding