RNA Biosynthesis(Transcription)

1 ．复制和转录过程有什么相似之处？又各有什么特点？

2. 试述真核细胞 RNA 转录后的加工修饰

Concept The process of copying DNA to RNA by an enzyme called

RNA polymerase (RNAP)

The transfer of genetic information from DNA into RNA

transcription

RNADNA

Content

Templates and Enzymes The Process of Transcription Post-transcriptional Modification

The difference between replication and transcription

Substances for transcription

Substrates: NTP (ATP, UTP, GTP, CTP)

Template: DNA

Enzyme: RNA polymerase( RNA-pol)

Other protein factors

Templates and Enzymes

Section I

Template for transcription

• Structural gene : a DNA fragment coding for any

RNA or protein product other than a regulatory

element

• Template strand (sense strand or Watson strand) :

the strand of DNA that the RNA polymerase uses as

a template to produce complementary mRNA

• Coding strand (antisense strand or Crick strand) :

the strand that is not used as the template

it has the same base sequence as the RNA transcript p

roduced (although with thymine replaced by uracil)

5′···GCAGTACATGTC ···3′

3′··· c g t g a t g t a c a g ···5′

5′···GCAGUACAUGUC ···3′

N······Ala · Val · His · Val ······C

Coding stand

Template strand

mRNA

Protein

transcription

translation

5

3

3

5

Template strandCoding strand

Coding strandTemplate strand

Structural gene

Direction of transcription

Direction of transcription

Asymmetric transcription

• certain region of only one strand of DNA

serves as a template for transcription

• The template stand is not always restricted to

the same single DNA strand

RNA polymeraseProkaryotic RNA polymerase

core enzyme holoenzyme

The binding site of RNApol holoenzyme on the transcription initiation region

Eukaryotic RNA polymerase

种类 Ⅰ Ⅱ Ⅲ

对鹅膏蕈碱的反应

45S-rRNA hnRNA 5S-rRNA

tRNA

snRNA

耐受 极敏感 中度敏感

转录产物

A site for RNA pol to identify and bind on the template

Operon: a unit of transcription in prokaryotes in

cluding one or more structural genes and upstream regul

on

53

35

Structural generegulon

RNA-pol

promoter : the site on the template DNA for RNA pol binding

RNA polymerase protectionassa

y

目 录

Transcription initiation

T T G A C AA A C T G T

-35 region

(Pribnow box)

T A T A A T Pu A T A T T A Py

-10 region

1-30-50 10-10-40 -205

3

3

5

Prokaryotic promote conservative sequence

recognition site

55

RNA pol protection region Structural gene

33

TATA bax

CAAT bax

GC bax

enhancer

Cis-actin element

Structural gene-GCGC---CAAT---TATA

Transcription initiation

Eukaryotic promote conservative sequence

The Process of Transcription

Section II

Initiation

Two issues required to be addressed

the binding of RNA polymerase to the initial region of the transcription template precisely

• only one strand of DNA, the template strand, is used to produce mRNA

Prokaryotic transcription

• The DNA is unwound and becomes single-stranded in the vicinity of the initiation site (defined as +1).

•RNA pol holoenzyme (2) specifically binding to

the promoter region on DNA

• 1st poly reaction catalyzed by RNA pol and transcription init

iation complex is formed

RNApol (2) - DNA - pppGpN- OH 3

transcription initiation complex

5-pppG -OH + NTP 5-pppGpN - OH 3 + ppi

Initiation

Elongation

subunit dissociates from the holoenzyme

， and elongation proceeds

• with the effect of the core enzyme ， NTP maintain p

olymerization ， RNA strand elongated

(NMP) n + NTP (NMP) n+1 + PPi

transcription bubble ：

RNA-pol （ Core enzyme ） ···· DA ···· RNA 目 录

目 录

53

DNA

Feather phenomena during prokaryotic transcription

ribosome

RNA

RNA pol

Rho (ρ) factor dependent transcription termination

Rho-independent transcription termination

Termination

The process of the RNA polymerase stopping on

DNA template and the dissociation of the RNA from the

transcription complex

Classification

A T P

Rho (ρ) factor dependent transcription termination

Termination independent on Rho factor

The terminator sequence within the RNA for the stop

of RNA polymerase. The terminator sequence usually for

ms a stem-loop hairpin structure leading to the dissociation

of the RNAP from the DNA template

5`UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU... 3` 5`UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU... 3`

RNA

5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT... 3 DNA

UUUU...…

UUUU...…

5`UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU... 3`

stem-loop/hairpin structure

Mechanism involved in the termination by stem loop

•allosteric change of RNA poly for the stop of transcription

• dissociation of transcription complex for the release of RNA

5´pppG

5 3

35

RNA-pol

Eukaryotic transcription initiation

initiation

•A variety of upstream control elements exist

•RNA-pol does not directly bind to the template in

the initiation

•Much more complicated than prokaryotic transcr

iption

TranscriptionInitiation n site

TATA box

CAAT box

GC box

enhancer

cis-acting element

The upstream region from the transcription initiation

AATAAA

Excision andadding tail

Transcription

Termination site

Modification site

exonTranslation initiation site

intron

OCT-1

OCT-1 ： ATTTGCAT octamer

Transcription factor

Trans-acting factors :The proteins with ability to recognize and bind directly or indirectly

to the upstream control element of DNA

Hundreds factors have been reported

Transcriptional factors (TF): Among the trans-acting factors, the proteins with ability to bind

directly or indirectly to RNA pol

参与 RNA-polⅡ转录的 TFⅡ

蛋白激酶活性，使CTD磷酸化

TFⅡ H

ATPase57（） 34（）TFⅡ E

解螺旋酶30，74TFⅡ F

促进RNA-polⅡ 结合及作为其他因子结合的桥梁

33TFⅡ B

稳定TFⅡ D-DNA复合物12，19，35TFⅡ A

辅助TBP-DNA结合TAF**

结合TATA盒TBP* 38TFⅡ D

功 能亚基组成，分子量(kD)转录因子

蛋白激酶活性，使CTD磷酸化

TFⅡ H

ATPase57（） 34（）TFⅡ E

解螺旋酶30，74TFⅡ F

促进RNA-polⅡ 结合及作为其他因子结合的桥梁

33TFⅡ B

稳定TFⅡ D-DNA复合物12，19，35TFⅡ A

辅助TBP-DNA结合TAF**

结合TATA盒TBP* 38TFⅡ D

功 能亚基组成，分子量(kD)转录因子

Pre-initiation complex ( PIC)

Eukaryotic transcription initiation require a variety of transcription factor because of the indirectly binding of Eukaryotic RNA-pol to DNA

POL-ⅡTF FⅡ

ⅡA

ⅡB

PIC transcription catalyzed by RNA-Pol Ⅱ

POL-ⅡTF FⅡ ⅡH ⅡE

TBP TAF

TF D- A- B-DNA complexⅡ Ⅱ Ⅱ

TATA

ⅡA

ⅡBTBP TAF

TATA

ⅡHⅡE

CTD- P

After PIC assemble ， CTD phosphorylation by TF HⅡ

Piecing theory

The eukaryotic transcription requires 3 to 5

TF binding together and becoming the complex

for the activation of specific gene transcription

Elongation

•Similar to prokaryotic transcription elongation.

•synchronous transcription and

translation is not observed because of segregation of

nuclear membrane• The forward moving process of RNA-pol encountering nucleosome

•The translocation and dissociation of nucleosomes observed during the transcription

RNA-Pol

RNA-Pol

RNA-Pol

nucleosomeNucleosome translocation during elongation

Direction of transcription

5------AAUAAA-

5 ------AAUAAA--

nuclease-GUGUGUG

RNA-polAATAAA GTGTGTG

Modification site for

transcription termination

55

33

3 adding tail

AAAAAAA······ 3 mRNA

Termination

——associated with post-transcriptional modification

Post-transcriptional modification

Sction III

Concept

In eukaryotic cells, a genetic process for the conversion of a primary transcript RNA to mature RNA

Major modifications

splicing cleavage

modification addition

Eukaryotic mRNA processing

Modifications to the “head" (5') and “tail" (3') ends of the mRNA

• 5 capping (m7GpppGp —)

• 3 polyadenylation (poly A tail)

5 cap structure

5 pppGp…

5 GpppGp…

pppG

ppi

GMP

transferase

5 m7GpppGp…

methyltransferase SAM

The formation of 5 cap

5 ppGp…phosphatase

Pi

mRNA splicing

• hetero-nuclear RNAa( hnRNA) : the primary mRNA

within nucleus

• snRNA: a class of small RNA molecules within the nu

cleus

Proteins within Nuclear

small nuclear ribonucleoproteins （ splicesome ）

snRNA

a strand of DNA containing both introns and exons.

Most advanced eukaryotes have interrupted genes and intro

ns are longer than exons. The resulting gene is much longer

than its coding region

Interrupted gene

CA B D

Coding region A 、 B 、 C 、 D

Non-coding region

Exon and intron

• exon the regions of a gene that are represented

in mature form of the messenger RNA (mRNA)

• intronThe sections of a gene that are present in p

recursor mRNA (pre-mRNA) and removed by a

process called splicing during the processing to

mature RNA

Chicken Ovalbumin Gene

hnRNA

Modifications to the "front" and "back" ends

hnRNA splicing

Mature mRNA

Chicken Ovalbumin Gene and its Transcription and Post-transcriptional Modification 目 录

Electron-microscopic picture of the mature mRNA and DNA hybridization of Chicken Ovalbumin

DNA

mRNA

目 录

Classification of introns

Four classes: according to gene types and splicing methods

I ： mainly found in mitochondria, chloroplast, and c

ertain low-level eukaryotic rRNA genes

II ： found in mitochondria, chloroplast, in mRNA g

enes

III ： frequently found in the splicing after loop for

mation ， in most mRNA genes

IV ： tRNA genes and primary transcription product

ion intron ， splicing requiring enzyme and ATP

mRNA splicing to remove the introns of hnRNA and ligate the exons

Splicesome is formed by ligation of snRNP and hnRNA

目 录

①

②

③

UACUACA - AG

UG

U4

U5

U6

E1

E2

U1 U2

UACUACA - AG

UGU6

E1

E2

U1 、 U4 、 U5

pG-OH(ppG-OH, pppG-OH)

U-OH

GpUpGpA

1ST transesterification reaction

2nd transesterification reaction

UpA GpUExon 1 Intron Exon 2

G-OH

UpU

pGpA

•剪接过程的二次转酯反应

Differential RNA processing: RNA editing shows that the

coding sequence of genes has a variety of differential function after post-transcription processing

mRNA editing

Human apo B gene

mRNA （ 14500 Nucleotides ）

liverapo B100

（ MW 500 000 ）intestinal cells

apo B48（ MW 240 000 ）

mRNA editing

Post-transcriptional process of tRNA

tRNA precursor

RNA pol Ⅲ

TGGCNNAGTGC GGTTCGANNCC

DNA

目 录

RNAase P 、 endonuclease

目 录

tRNA nucleotide transferase 、 ligase

ATP

ADP

目 录

Base modifications

•Reduced reaction

e.g. ： U DHU• Translocation within nucleotides

e.g. ： U ψ

•Deamination reaction

e.g. ： A I

e.g. ： A Am

•Methylation

（ 1 ）（ 1 ）

（ 3 ）

（ 2 ）

（ 4 ）

目 录

transcription

45S - rRNA

splice

18S - rRNA 5.8S and 28S-rRNA

rDNAintron intron 28S5.8S18S

Post-transcriptional process of rRNA

an RNA molecule that catalyzes a chemical reaction

Ribozyme

Secondary structure of Tetrahymena rRNA intron

Self-splicing rRNA of Tetrahymena

5´-terminal sequence

• hammerhead structure —— The most simple secondary structure of ribozyme

substrate

• Around 60 nucleotides

• With catalytic part and

substrate in the same molecular

• hammerhead structure

consisted of catalytic part and

substrate

• Apart from rRNA ， the process of tRNA 、 mRNA

also exists Self-splicing

Significance of ribozyme research

The important supplementary to the central dogma

The challenge to traditional enzymology

Design a ribozyme according to the native ribozyme s

tructure

The designed ribozyme

• Synthetic nucleic acids shown with thick lines

• Native nucleic acids shown with thin lines

• consensus sequence shown with

• Spliced site shown by arrow

目 录

Appendix

G

OH

3´

G

5´

OH

5´

3´

G

OH

414

G

399

5´

3´395

5´

3´

E1

E2

I

Self-splicing RNA of Tetrahymena

5´

3´

L19RNA

Fragment with catalytic activity