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Chapter 11
Transcription
The biochemistry and molecular
biology department of CMU
Transcription
The synthesis of RNA molecules using
DNA strands as the templates so that
the genetic information can be
transferred from DNA to RNA.
Similarity between
replication and transcription
• Both processes use DNA as the
template.
• Phosphodiester bonds are formed in
both cases.
• Both synthesis directions are from 5´
to 3´.
Differences between
replication and transcription
replication
transcription
template
double strands
single strand
substrate
dNTP
NTP
primer
yes
no
Enzyme
DNA polymerase
RNA polymerase
product
dsDNA
ssRNA
base pair
A-T, G-C
A-U, T-A, G-C
Section 1
Template and Enzymes
• The whole genome of DNA needs to
be replicated, but only small portion
of genome is transcribed in response
to the development requirement,
physiological need and
environmental changes.
• DNA regions that can be transcribed
into RNA are called structural genes.
§1.1 Template
The template strand is the strand
from which the RNA is actually
transcribed. It is also termed as
antisense strand.
The coding strand is the strand
whose base sequence specifies the
amino acid sequence of the encoded
protein. Therefore, it is also called as
sense strand.
5'
GCAGTACATGTC
3' coding
3'
CGTCATGTACAG
5'
strand
template
strand
transcription
5'
GCAGUACAUGUC
3'
RNA
Asymmetric transcription
• Only the template strand is used for the
transcription, but the coding strand is
not.
• Both strands can be used as the
templates.
• The transcription direction on different
strands is opposite.
• This feature is referred to as the
asymmetric transcription.
5'
3'
3'
5'
Organization of coding information in
the adenovirus genome
§1.2 RNA Polymerase
• The enzyme responsible for the RNA
synthesis is DNA-dependent RNA
polymerase.
– The prokaryotic RNA polymerase is a
multiple-subunit protein of ~480kD.
– Eukaryotic systems have three kinds of
RNA polymerases, each of which is a
multiple-subunit protein and responsible
for transcription of different RNAs.
Holoenzyme
The holoenzyme of RNA-pol in E.coli
consists of 5 different subunits: 2  
.
holoenzyme
core enzyme





RNA-pol of E. Coli
subunit
MW
function

36512
Determine the DNA to be
transcribed

150618
Catalyze polymerization

155613
Bind & open DNA template
70263
Recognize the promoter
for synthesis initiation

• Rifampicin, a therapeutic drug for
tuberculosis treatment, can bind
specifically to the  subunit of RNApol, and inhibit the RNA synthesis.
• RNA-pol of other prokaryotic
systems is similar to that of E. coli in
structure and functions.
RNA-pol of eukaryotes
RNA-pol
I
II
III
products
45S rRNA
hnRNA
5S rRNA
tRNA
snRNA
Sensitivity
to Amanitin
No
high
moderate
Amanitin is a specific inhibitor of RNA-pol.
§1.3 Recognition of Origins
• Each transcriptable region is called
operon.
• One operon includes several structural
genes and upstream regulatory
sequences (or regulatory regions).
• The promoter is the DNA sequence that
RNA-pol can bind. It is the key point
for the transcription control.
Promoter
regulatory
sequences
5'
3'
promotor
RNA-pol
structural gene
3'
5'
Prokaryotic promoter
3'
5'
-50
3'
-40
-30
-20
-35
region
TTGACA
AACTGT
-10
1
-10
region
TATAAT
ATATTA
(Pribnow box)
Consensus sequence
10
start
5'
Consensus Sequence
Frequency in 45 samples
38 36 29
37 37 28
40 25 30
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
• Three phases: initiation, elongation,
and termination.
• The prokaryotic RNA-pol can bind to
the DNA template directly in the
transcription process.
• The eukaryotic RNA-pol requires cofactors to bind to the DNA template
together in the transcription process.
§2.1 Transcription of Prokaryotes
• Initiation phase: RNA-pol recognizes
the promoter and starts the
transcription.
• Elongation phase: the RNA strand is
continuously growing.
• Termination phase: the RNA-pol stops
synthesis and the nascent RNA is
separated from the DNA template.
a. Initiation
• RNA-pol recognizes the TTGACA
region, and slides to the TATAAT
region, then opens the DNA duplex.
• The unwound region is about 171 bp.
• The first nucleotide on RNA transcript
is always purine triphosphate. GTP is
more often than ATP.
• The pppGpN-OH structure remains on
the RNA transcript until the RNA
synthesis is completed.
• The three molecules form a
transcription initiation complex.
RNA-pol (2) - DNA - pppGpN- OH 3
• No primer is needed for RNA
synthesis.
• The  subunit falls off from the RNApol once the first 3,5 phosphodiester
bond is formed.
• The core enzyme moves along the
DNA template to enter the elongation
phase.
b. Elongation
• The release of the  subunit causes
the conformational change of the
core enzyme. The core enzyme slides
on the DNA template toward the 3
end.
• Free NTPs are added sequentially to
the 3 -OH of the nascent RNA strand.
(NMP)n + NTP
RNA strand
substrate
(NMP)n+1 + PPi
elongated
RNA strand
• RNA-pol, DNA segment of ~40nt and
the nascent RNA form a complex
called the transcription bubble.
• The 3 segment of the nascent RNA
hybridizes with the DNA template, and
its 5 end extends out the
transcription bubble as the synthesis
is processing.
Transcription bubble
RNA-pol of E. Coli
RNA-pol of E. Coli
Simultaneous
transcriptions and
translation
c. Termination
• The RNA-pol stops moving on the
DNA template. The RNA transcript
falls off from the transcription
complex.
• The termination occurs in either  dependent or  -independent manner.
The termination function of  factor
The  factor, a hexamer, is a ATPase
and a helicase.
-independent termination
• The termination signal is a stretch of
30-40 nucleotides on the RNA
transcript, consisting of many GC
followed by a series of U.
• The sequence specificity of this
nascent RNA transcript will form
particular stem-loop structures to
terminate the transcription.
DNA
rplL protein
5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGGCACCAGCCTTTTT... 3
5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT... 3
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 RNARNA 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
enhancer
GC box
CAAT box
(Hogness 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)
• TBP of TFII D binds TATA
• TFII A and TFII B bind TFII D
• TFII F-RNA-pol complex binds TFII B
• TFII F and TFII E open the dsDNA
(helicase and ATPase)
• TFII H: completion of PIC
Pre-initiation complex (PIC)
RNA pol II
TF II F
TF II
A
TBP TAF
TATA
TF II
B
TF II E
TF II H
DNA
Phosphorylation of RNA-pol
• TF II H is of protein kinase activity to
phosphorylate CTD of RNA-pol. (CTD
is the C-terminal domain of RNA-pol)
• Only the p-RNA-pol can move toward
the downstream, starting the
elongation phase.
• Most of the TFs fall off from PIC
during the elongation phase.
b. Elongation
• The elongation is similar to that of
prokaryotes.
• The transcription and translation do
not take place simultaneously since
they are separated by nuclear
membrane.
nucleosome
RNA-Pol
moving
direction
RNA-Pol
RNA-Pol
c. Termination
• The termination sequence is AATAAA
followed by GT repeats.
• The termination is closely related to
the post-transcriptional modification.
Section 3
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
H2N
O
N
N
O
O
O
NH
5'
H2C O P O P O P O CH2
5'
HN
O
O
N
O
O
N
NH2
N
O
CH3
Pi
O
O
P O
O
m7GpppGp----
OH
AAAAA-OH
3'
ppp5'NpNp
Pi
removing
phosphate group
pp5'NpNp
GTP forming 5'-5'
PPi
triphosphate group
G5'ppp5'NpNp
methylating at G7
m
7
GpppNpNp
methylating at C2' of the
first and second
nucleotides after G
m
7
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
The matured mRNAs are much shorter than
the DNA templates.
Split gene
The structural genes are composed of
coding and non-coding regions that
are alternatively separated.
7 700 bp
L
1
A
2
3
B C D
4
5
E
6
F
7
G
A~G no-coding region 1~7 coding region
Exon and intron
Exons are the coding sequences that
appear on split genes and primary
transcripts, and will be expressed to
matured mRNA.
Introns are the non-coding sequences
that are transcripted into primary
mRNAs, and will be cleaved out in the
later splicing process.
mRNA splicing
Splicing mechanism
lariat
Twice transesterification
intron
5'
5'exon
3'exon
U pA
G pU
3'
first transesterification
pG-OH
pGpA
5'
UOH
3'
G pU
second transesterification
5' pGpA
5'
U pU
3'
GOH 3'
d. mRNA editing
• Taking place at the transcription
level
• One gene responsible for more than
one proteins
• Significance: gene sequences, after
post-transcriptional modification,
can be multiple purpose
differentiation.
Different pathway of apo B
Human apo B
gene
hnRNA (14 500 base)
CAA to UAA
At 6666
liver
apo B100
（500 kD）
intestine
apo B48
（240 kD）
§3.2 Modification of tRNA
Precursor transcription
DNA
TGGCNNAGTGC
GGTTCGANNCC
RNA-pol III
tRNA precursor
Cleavage
RNAase P
endonuclease
ligase
Addition of CCA-OH
tRNA nucleotidyl
transferase
ATP
ADP
Base modification
（2）
（1）
（1）
1. Methylation
A→mA, G→mG
2. Reduction
U→DHU
3. Transversion
U→ψ
（3）
（4）
4. Deamination
A→I
§3.3 Modification of rRNA
• 45S transcript in nucleus is the
precursor of 3 kinds of rRNAs.
• The matured rRNA will be assembled
with ribosomal proteins to form
ribosomes that are exported to
cytosolic space.
rRNA
18S
5.8S
28S
45S-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
• Be a supplement to the central
dogma
• Redefine the enzymology
• Provide a new insights for the origin
of life
• Be useful in designing the artificial
ribozymes as the therapeutical
agents
Artificial
ribozyme
• Thick lines:
artificial ribozyme
• Thin lines:
natural ribozyme
• X: consensus
sequence
• Arrow: cleavage
point