Transcript RNA Biosynthesis (Transcription)

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
DNA
RNA
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
Section I
Templates and Enzymes
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
produced (although with thymine replaced by
uracil)
5′···GCAGTACATGTC ···3′
Coding stand
3′··· c g t g a t g t a c a g ···5′
Template strand
transcription
5′···GCAGUACAUGUC ···3′
mRNA
translation
N······Ala · Val · His · Val ······C Protein
Structural gene
Direction of
transcription
5
Coding strand
Template strand
3
3
Template strand
Coding strand
5
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 polymerase
Prokaryotic RNA polymerase
core enzyme
holoenzyme









The binding site of RNApol holoenzyme on the
transcription initiation region
Eukaryotic RNA polymerase
种类
Ⅰ
Ⅱ
Ⅲ
转录产物
45S-rRNA
hnRNA
5S-rRNA
tRNA
对鹅膏蕈碱
的反应
耐受
极敏感
snRNA
中度敏感
A site for RNA pol to identify and bind on the template
Operon:
a unit of transcription in prokaryotes
including one or more structural genes and upstream
regulon
regulon
5
3
Structural gene
RNA-pol
promoter : the site on the template DNA for RNA pol binding
3
5
RNA
polymerase
protectionassay
目录
RNA pol protection region
Structural gene
3
5
5
3
5
3
-50
-40
-30
-20
-10
1
10
3
5
-35 region
Transcription
initiation
-10 region
TTGACA
AA C T G T
T A T A A T Pu A
T A T T A Py
recognition site
Prokaryotic promote conservative
sequence
(Pribnow box)
Cis-actin element
Structural gene
-GCGC---CAAT---TATA
Transcription
initiation
enhancer
TATA bax
CAAT bax
GC bax
Eukaryotic promote conservative sequence
Section II
The Process of Transcription
Prokaryotic transcription
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
Initiation
•RNA pol holoenzyme (2) specifically binding to
the promoter region on DNA
• The DNA is unwound and becomes single-stranded in the
vicinity of the initiation site (defined as +1).
• 1st poly reaction catalyzed by RNA pol and transcription
initiation complex is formed
5-pppG -OH + NTP  5-pppGpN - OH 3 + ppi
transcription initiation complex
RNApol (2) - DNA - pppGpN- OH 3
Elongation
•  subunit dissociates from the holoenzyme
，and elongation proceeds
•
with the effect of the core enzyme，NTP maintain
polymerization，RNA strand elongated
(NMP) n + NTP  (NMP) n+1 + PPi
transcription bubble：
RNA-pol （Core enzyme ） ···· DA ···· RNA
目录
目录
DNA
5
RNA
3
RNA pol
ribosome
Feather phenomena during prokaryotic
transcription
Termination
The process of the RNA polymerase stopping on DNA
template and the dissociation of the RNA
from the
transcription complex
Classification

Rho (ρ) factor dependent transcription termination

Rho-independent transcription termination
Rho (ρ) factor dependent transcription termination
ATP
Termination independent on Rho factor
The terminator sequence within the RNA for the stop
of RNA polymerase. The terminator sequence usually
forms a stem-loop hairpin structure leading to the
dissociation of the RNAP from the DNA template
DNA
5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT... 3
5`UUGCAGCCUGACAAAUCAGGCUGAUGGCUGGUGACUUUUUAGUCACCAGCCUUUUU... 3`
RNA
UUUU...…
UUUU...…
stem-loop/hairpin structure
RNA-pol
5
3
3
5
5´pppG
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
Eukaryotic transcription initiation
initiation
•
A variety of upstream control elements exist
•
RNA-pol does not directly bind to the template in
the initiation
•
Much
transcription
more
complicated
than
prokaryotic
The upstream region from the transcription initiation
Modification
site
cis-acting element
Excision and
adding tail
AATAAA
Translation
initiation site
Transcription
Initiation n site
enhancer
exon
intron
Transcription
Termination site
TATA box
OCT-1
CAAT box
GC box
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
转录因子 亚基组成，分子量(kD)
TFⅡD
TBP* 38
TAF**
TFⅡA
12，19，35
TFⅡB
33
TFⅡF
TFⅡE
TFⅡH
30，74
57（） 34（）
功 能
结合TATA盒
辅助TBP-DNA结合
稳定TFⅡD-DNA复合物
促进RNA-polⅡ结合及作
为其他因子结合的桥梁
解螺旋酶
ATPase
蛋白激酶活性，使CTD磷
酸化
Pre-initiation complex ( PIC)
Eukaryotic transcription initiation require
a variety of transcription factor because of the
indirectly binding of Eukaryotic RNA-pol to
DNA
PIC transcription catalyzed by RNA-Pol Ⅱ
TBP TAF
TATA ⅡB
ⅡA
TFⅡD-ⅡA-ⅡB-DNA complex
POL-Ⅱ
TFⅡF
ⅡH
ⅡE
ⅡE
ⅡH
TAF
TBP
POL-Ⅱ
TFⅡFTATA ⅡB
ⅡA
CTD- P
After PIC assemble，CTD phosphorylation by TFⅡH
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
Nucleosome translocation during elongation
nucleosome
RNA-Pol
Direction of
transcription
RNA-Pol
RNA-Pol
Termination
——associated with post-transcriptional modification
AAAAAAA······ 3
mRNA
3 adding tail
nuclease
3
5
5
AATAAA
GTGTGTG
Modification site for
transcription termination
RNA-pol
3
Sction III
Post-transcriptional modification
Concept
In eukaryotic cells, a genetic process for
the conversion of a primary transcript
RNA to mature RNA
Major modifications
splicing
modification
cleavage
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
The formation of 5 cap
5 pppGp…
phosphatase
5 ppGp…
Pi
pppG
GMP
transferase
ppi
5 GpppGp…
SAM
methyltransferase
5 m7GpppGp…
mRNA splicing
• hetero-nuclear RNAa( hnRNA) : the primary mRNA
within nucleus
• snRNA: a class of small RNA molecules within the
nucleus
snRNA
Proteins within Nuclear
small nuclear ribonucleoproteins
（splicesome）
Interrupted gene
a strand of DNA containing both introns and exons.
Most advanced eukaryotes have interrupted genes and
introns are longer than exons. The resulting gene is much
longer than its coding region
A
B
Coding region A、B、C、D
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)
• intron
The sections of a gene that are present in
precursor 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 Posttranscriptional Modification
目录
mRNA
DNA
Electron-microscopic picture of the mature mRNA and DNA
hybridization of Chicken Ovalbumin
目录
Classification of introns
Four classes: according to gene types and splicing methods
I：mainly found in mitochondria, chloroplast, and
certain low-level eukaryotic rRNA genes
II： found in mitochondria, chloroplast, in mRNA
genes
III： frequently found in the splicing after loop
formation ，in most mRNA genes
IV：tRNA genes and primary transcription
production 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
①
目录
E1
②
U6 U4
U5
UG
UACUACA - AG
U1
E2
U2
U1、U4、U5
E1
③
U6 UG
UACUACA - AG
E2
Intron
Exon 1
Exon 2
UpA
GpU
pG-OH
(ppG-OH, pppG-OH)
1ST transesterification
reaction
U-OH
•剪接
过程的
二次转
酯反应
pGpA
GpU
2nd transesterification
reaction
pGpA
G-OH
UpU
mRNA editing
Human apo B gene
mRNA（14500 Nucleotides）
mRNA editing
liver
apo B100
（MW 500 000）
intestinal cells
apo B48
（MW 240 000）
Differential RNA processing: RNA editing shows that the
coding sequence of genes has a variety of differential function after
post-transcription processing
Post-transcriptional process of tRNA
DNA
TGGCNNAGTGC
GGTTCGANNCC
RNA pol Ⅲ
tRNA
precursor
目录
RNAase P、
endonuclease
目录
tRNA nucleotide
transferase 、ligase
ATP
ADP
目录
Base modifications
•Methylation
（1）
（2）
（1）
e.g.：A  Am
•Reduced reaction
e.g.：U  DHU
• Translocation within nucleotides
e.g.：U  ψ
（3）
•Deamination reaction
（4）
e.g.：A  I
目录
Post-transcriptional process of rRNA
rDNA
18S
intron
5.8S
intron
28S
transcription
45S - rRNA
splice
18S - rRNA
5.8S and 28S-rRNA
Ribozyme
an RNA molecule that catalyzes a
chemical reaction
Self-splicing rRNA of Tetrahymena
Secondary structure of Tetrahymena rRNA intron
5´-terminal sequence
• Apart from rRNA，the process of tRNA、mRNA
also exists Self-splicing
• hammerhead structure —— The most simple secondary
structure of ribozyme
• Around 60 nucleotides
substrate
• With catalytic part and
substrate in the same molecular
• hammerhead structure
consisted of catalytic part and
substrate
Significance of ribozyme research

The important supplementary to the central dogma

The challenge to traditional enzymology

Design a ribozyme according to the native ribozyme
structure
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
5´
5´
E1
5´
Self-splicing RNA
of Tetrahymena
G
OH
414
G
OH
I
G
3´
E2
3´
5´
3´
5´
OH
G
399
L19
RNA
Fragment with
catalytic activity
395
3´
3´