Transcription

Transcription
Tapeshwar Yadav
(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry

• The flow of information in the cell starts at
DNA, which replicates to form more DNA.
Information is then ‘transcribed” into
RNA, and then it is “translated” into
protein.
• Information does not flow in the other
direction.
• A few exceptions to the Central Dogma
exist
• some RNA viruses, called “retroviruses”.

The synthesis of RNA molecules using
DNA strands as the templates so that
the genetic information can be
transferred from DNA to RNA.
Transcription

• 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

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

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.

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'

§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.

core enzymeholoenzyme
Holoenzyme
The holoenzyme of RNA-pol in E.coli
consists of 5 different subunits: α2 β β′
ωσ.
ω
β′
β
αα
σ

subunit MW function
α 36512
Determine the DNA to be
transcribed
β 150618 Catalyze polymerization
β′ 155613 Bind & open DNA template
σ 70263
Recognize the promoter
for synthesis initiation
RNA-pol of E. Coli

• Rifampicin, a therapeutic drug for
tuberculosis treatment, can bind
specifically to the β 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.

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.

• 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

5'
3'
3'
5'
regulatory
sequences
structural gene
promotorRNA-pol
Promoter

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

• 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 co-
factors 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 (α2ββ′σ) - DNA - pppGpN- OH 3′

• No primer is needed for RNA
synthesis.
• The σ 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.

b. Elongation
• The release of the σ 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.

• 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.

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 ρ
-dependent or ρ -independent
manner.

The termination function of ρ factor
The ρ factor, a hexamer, is a ATPase
and a helicase.

ρ-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.

• 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

§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

structural gene
GCGC CAAT TATA
intronexon exon
start
CAAT box
GC box
enhancer
cis-acting element
TATA box (Hogness box)
Cis-acting element

• 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

TF for eukaryotic transcription

• 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)

Pre-initiation complex (PIC)
RNA pol II
TF II F
TBP TAF
TATA
DNA
TF II
A
TF II
B
TF II E
TF II H

• 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

• 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

RNA-Pol
RNA-Pol
RNA-Pol
nucleosome
moving
direction

• The termination sequence is
AATAAA followed by GT repeats.
• The termination is closely related to
the post-transcriptional modification.
c. Termination

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.

• 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

CH3
O
O OH
CH2
PO
O
O
N
NH
N
N
O
NH2
AAAAA-OH
O
Pi
5'
3'
O
OHOH
H2C
N
HN
N
N
O
H2N O P
O
O
O P
O
O
O P
O
O
5'
a. Capping at the 5′- end
m7
GpppGp----

• 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.

The matured mRNAs are much shorter than
the DNA templates.
DNA
mRNA
c. mRNA splicing

A~G no-coding region 1~7 coding region
L 1 2 3 4 5 6 7
7 700 bp
The structural genes are composed of
coding and non-coding regions that
are alternatively separated.
Split gene
EA B C D F G

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.

• 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

RNAase P
endonuclease
Cleavage
ligase

tRNA nucleotidyl
transferase
ATP ADP
Addition of CCA-OH

Base modification
（ 1 ）
（ 1 ）
（ 3 ）
（ 2 ）
（ 4 ）
1. Methylation
A→mA, G→mG
2. Reduction
U→DHU
3. Transversion
U→ψ
4. Deamination
A→I

Transcription

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