Transcriptionlibvolume1.xyz/biochemistry/bsc/semester5/... · –The prokaryotic RNA polymerase is...
Transcript of Transcriptionlibvolume1.xyz/biochemistry/bsc/semester5/... · –The prokaryotic RNA polymerase is...
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Chapter 11
Transcription
The biochemistry and molecular
biology department of CMU
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The synthesis of RNA molecules using
DNA strands as the templates so that
the genetic information can be
transferred from DNA to RNA.
Transcription
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• Both processes use DNA as the
template.
• Phosphodiester bonds are formed in
both cases.
• Both synthesis directions are from 5´́́́
to 3´́́́.
Similarity between
replication and transcription
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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
Differences between
replication and transcription
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Section 1
Template and Enzymes
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• 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.
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§§§§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.
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G C A G T A C A T G T C5' 3'
3' C G T C A T G T A C A G 5' template strand
coding strand
transcription
RNAG C A G U A C A U G U C5' 3'
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• 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.
Asymmetric transcription
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5'
3'
3'
5'
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Organization of coding information in the adenovirus genome
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§§§§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.
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core enzymeholoenzyme
Holoenzyme
The holoenzyme of RNA-pol in E.coli
consists of 5 different subunits: αααα2 ββββ β′β′β′β′
ωωωωσσσσ.
ωωωω
β′β′β′β′
ββββ
αααα αααα
σσσσ
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subunit MW function
αααα 36512 Determine the DNA to be
transcribed
ββββ 150618 Catalyze polymerization
β′β′β′β′ 155613 Bind & open DNA template
σσσσ 70263 Recognize the promoter
for synthesis initiation
RNA-pol of E. Coli
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• Rifampicin, a therapeutic drug for
tuberculosis treatment, can bind
specifically to the ββββ subunit of RNA-
pol, and inhibit the RNA synthesis.
• RNA-pol of other prokaryotic
systems is similar to that of E. coli in
structure and functions.
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RNA-pol I II III
products 45S rRNA hnRNA
5S rRNA
tRNA
snRNA
Sensitivity
to Amanitin No high moderate
RNA-pol of eukaryotes
Amanitin is a specific inhibitor of RNA-pol.
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• 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.
§§§§1.3 Recognition of Origins
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5'
3'
3'
5'
regulatory sequences
structural gene
promotorRNA-pol
Promoter
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5'
3'
3'
5'-50 -40 -30 -20 -10 1 10
start -10
region
T A T A A T
A T A T T A
(Pribnow box)
-35 region
T T G A C A A A C T G T
Prokaryotic promoter
Consensus sequence
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Consensus Sequence
Frequency in 45 samples 38 36 29 40 25 30
37 37 28 41 29 44
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• 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.
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Section 2
Transcription Process
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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 co-
factors to bind to the DNA template
together in the transcription process.
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§§§§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.
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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.
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• 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 (αααα2ββ′σββ′σββ′σββ′σ) - DNA - pppGpN- OH 3′′′′
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• No primer is needed for RNA
synthesis.
• The σσσσ subunit falls off from the RNA-
pol once the first 3′′′′,5′′′′ phosphodiester
bond is formed.
• The core enzyme moves along the
DNA template to enter the elongation
phase.
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b. Elongation
• The release of the σσσσ 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.
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• 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.
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Transcription bubble
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RNA-pol of E. Coli
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RNA-pol of E. Coli
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Simultaneous
transcriptions and
translation
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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 ρρρρ -dependent or ρρρρ -independent manner.
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The termination function of ρρρρ factor
The ρρρρ factor, a hexamer, is a ATPase
and a helicase.
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ρρρρ-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.
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RNA
5′′′′TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGGCACCAGCCTTTTT... 3′′′′
DNA
UUUU...?
rplL protein
UUUU...?
5′′′′TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT... 3′′′′
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• 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.
Stem-loop disruption
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§§§§2.2 Transcription of Eukaryotes
• 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.
a. Initiation
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structural geneGCGC CAAT TATA
intronexon exon
start
CAAT box
GCbox
enhancer
cis-acting element
TATA box (Hogness box)
Cis-acting element
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TATA box
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• 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.
Transcription factors
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TF for eukaryotic transcription
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• 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)
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Pre-initiation complex (PIC)
RNA pol II
TF II F
TBP TAF
TATADNA
TF II
ATF II
B
TF II E
TF II H
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• 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.
Phosphorylation of RNA-pol
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• The elongation is similar to that of
prokaryotes.
• The transcription and translation do
not take place simultaneously since
they are separated by nuclear
membrane.
b. Elongation
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RNA-Pol
RNA-Pol
RNA-Pol
nucleosome
moving
direction
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• The termination sequence is AATAAA
followed by GT repeats.
• The termination is closely related to
the post-transcriptional modification.
c. Termination
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Section 3
Post-Transcriptional
Modification
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• 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.
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• 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
§§§§3.1 Modification of hnRNA
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CH3
O
O OH
CH2
PO
O
O
N
NHN
N
O
NH2
AAAAA-OH
O
Pi
5'
3'
O
OHOH
H2CN
HNN
N
O
H2N O P
O
O
O P
O
O
O P
O
O
5'
a. Capping at the 5′′′′- end
m7GpppGp----
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• 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.
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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.
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The matured mRNAs are much shorter than
the DNA templates.
DNA
mRNA
c. mRNA splicing
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A~G no-coding region 1~7 coding region
L 1 2 3 4 5 6 77 700 bp
The structural genes are composed of
coding and non-coding regions that
are alternatively separated.
Split gene
E A B C D F G
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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.
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mRNA splicing
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Splicing mechanism
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lariat
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U pA G pU5' 3'5'exon 3'exon
intron
pG-OH
pGpA
G pU 3'U5' OH
first transesterification
Twice transesterification
second transesterification
U5' pU 3'
pGpA
GOH
5'
3'
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• 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.
d. mRNA editing
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Different pathway of apo B
Human apo B
gene
hnRNA (14 500 base)
liver
apo B100
((((500 kD)))) intestine
apo B48
((((240 kD))))
CAA to UAA
At 6666
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§§§§3.2 Modification of tRNA
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tRNA precursor
RNA-pol III
TGGCNNAGTGC GGTTCGANNCC
DNA
Precursor transcription
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RNAase P
endonuclease
Cleavage
ligase
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tRNA nucleotidyl
transferase
ATP ADP
Addition of CCA-OH
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Base modification
((((1111))))
((((1111))))
((((3333))))
((((2222))))
((((4444))))
1. Methylation
A→mA, G→mG
2. Reduction
U→DHU
3. Transversion
U→ψ
4. Deamination
A→I
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§§§§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.
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rRNA
transcription
splicing
45S-rRNA
18S-rRNA5.8S and 28S-rRNA
28S5.8S18S
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• 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.
§§§§3.4 Ribozyme
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• Both the catalytic domain and the
substrate locate on the same
molecule, and form a hammer-head
structure.
• At least 13 nucleotides are conserved.
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Hammer-head
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• 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
Significance of ribozyme
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Artificial
ribozyme
• Thick lines: artificial ribozyme
• Thin lines: natural ribozyme
• X: consensus sequence
• Arrow: cleavage point