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Transcription
Transcription
replication transcription
primer yes no
transcription
holoenzyme
core enzyme
RNA-pol of E. Coli
subunit MW function
Determine the DNA to be
36512
transcribed
RNA-pol I II III
5S rRNA
products 45S rRNA hnRNA tRNA
snRNA
Sensitivity
No high moderate
to Amanitin
regulatory
structural gene
sequences
5' 3'
promotor
RNA-pol
3' 5'
Prokaryotic promoter
5' 3'
-50 -40 -30 -20 -10 1 10
3' 5'
-35
region -10 start
TTGACA region
AACTGT
TATAAT
ATATTA
(Pribnow box)
Consensus sequence
Consensus Sequence
Frequency in 45 samples 38 36 29 40 25 30
37 37 28 41 29 44
• The -35 region of TTGACA sequence
is the recognition site and the
binding site of RNA-pol.
• The -10 region of TATAAT is the
region at which a stable complex of
DNA and RNA-pol is formed.
Section 2
Transcription Process
General concepts
RNA
UUUU...…
UUUU...…
Stem-loop disruption
• The stem-loop structure alters the
conformation of RNA-pol, leading to
the pause of the RNA-pol moving.
• Then the competition of the RNA-
RNA hybrid and the DNA-DNA hybrid
reduces the DNA-RNA hybrid stability,
and causes the transcription
complex dissociated.
• Among all the base pairings, the
most unstable one is rU:dA.
§2.2 Transcription of Eukaryotes
a. Initiation
• Transcription initiation needs
promoter and upstream regulatory
regions.
• The cis-acting elements are the
specific sequences on the DNA
template that regulate the
transcription of one or more genes.
Cis-acting element
cis-acting element
structural gene
GCGC CAAT TATA
exon intron exon
start
TATA box (Hogness box)
GC box
TATA box
Transcription factors
• RNA-pol does not bind the promoter
directly.
• RNA-pol II associates with six
transcription factors, TFII A - TFII H.
• The trans-acting factors are the
proteins that recognize and bind
directly or indirectly cis-acting
elements and regulate its activity.
TF for eukaryotic transcription
Pre-initiation complex (PIC)
RNA pol II
TF II F TF II E
TF II TBP TAF
TF II
A TATA B
TF II H DNA
Phosphorylation of RNA-pol
RNA-Pol
moving
direction
RNA-Pol
RNA-Pol
c. Termination
Post-Transcriptional
Modification
• The nascent RNA, also known as
primary transcript, needs to be
modified to become functional tRNAs,
rRNAs, and mRNAs.
• The modification is critical to
eukaryotic systems.
§3.1 Modification of hnRNA
• Primary transcripts of mRNA are called as
heteronuclear RNA (hnRNA).
• hnRNA are larger than matured mRNA by
many folds.
• Modification includes
– Capping at the 5- end
– Tailing at the 3- end
– mRNA splicing
– RNA edition
a. Capping at the 5- end
OH OH
O
N
NH
O O O
O 5'
H2N N N H2C O P O P O P O CH2 N NH2
N
5' O
O O O
HN
N
O
CH 3
O OH
Pi
O P O AAAAA-OH 3'
O
m7GpppGp----
ppp5'NpNp
removing
Pi phosphate group
pp5'NpNp
GTP forming 5'-5'
triphosphate group
PPi
G5'ppp5'NpNp
methylating at G7
7
m GpppNpNp
methylating at C2' of the
first and second
nucleotides after G
7
m Gpppm2'Npm2'Np
• The 5- cap structure is found on
hnRNA too. The capping process
occurs in nuclei.
• The cap structure of mRNA will be
recognized by the cap-binding protein
required for translation.
• The capping occurs prior to the
splicing.
b. Poly-A tailing at 3 - end
• There is no poly(dT) sequence on the
DNA template. The tailing process
dose not depend on the template.
• The tailing process occurs prior to the
splicing.
• The tailing process takes place in the
nuclei.
c. mRNA splicing
mRNA
DNA
7 700 bp
L 1 2 3 4 5 6 7
A B C D E F G
5'exon 3'exon
5' U pA G pU 3'
first transesterification
pG-OH
pGpA
5' U OH G pU 3'
second transesterification
5' pGpA
5' U pU 3'
GOH 3'
d. mRNA editing
Human apo B
gene
DNA
TGGCNNAGTGC GGTTCGANNCC
RNA-pol III
tRNA precursor
Cleavage
RNAase P
endonuclease
ligase
Addition of CCA-OH
tRNA nucleotidyl
transferase
ATP ADP
Base modification
1. Methylation
(2) (1) A→mA, G→mG
(1)
2. Reduction
U→DHU
3. Transversion
U→ψ
(3) 4. Deamination
(4) A→I
§3.3 Modification of rRNA
transcription
splicing
18S-rRNA
5.8S and 28S-rRNA
§3.4 Ribozyme
• The rRNA precursor of tetrahymena
has the activity of self-splicing (1982).
• The catalytic RNA is called ribozyme.
• Self-splicing happened often for
intron I and intron II.
• Both the catalytic domain and the
substrate locate on the same
molecule, and form a hammer-head
structure.
• At least 13 nucleotides are conserved.
Hammer-head
Significance of ribozyme