Lecture 3
RNA Biosynthesis
(Transcription)
Transcription
•The synthesis of RNA molecules using
DNA strands as the templates so that the
genetic information can be transferred
from DNA to RNA.
Transcription
DNA
RNA
Similarity between
replication and transcription
• Template: both processes use DNA as
the template.
• Bond: phosphodiester bonds are
formed in both cases.
• Direction: 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(mRNA,tRNA,
rRNA)
base pair
A-T, G-C
A-U, T-A, G-C
Section 1
Template and Enzymes
§1.1 Template
• 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.
• Structural genes: DNA regions that can
be transcribed into RNA.
The template strand and the coding strand
•The template strand: the strand from which
the RNA is actually transcribed. It is also
termed as antisense strand.
•The coding strand: its sequence is the same
as the newly created RNA transcript (except
for the substitution of uracil for thymine). 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.
5'
3'
3'
5'
§1.2 RNA Polymerase
• The enzyme responsible for the RNA synthesis is
DNA-dependent RNA polymerase(DDRP).
– The prokaryotic RNA polymerase is a multiplesubunit protein of ~480kD.
– The holoenzyme of RNA-pol in E.coli consists
of 5 different subunits: 2   .
holoenzyme

Core enzyme

holoenzyme
core enzyme



RNA-pol of E. Coli
subunit
MW
function

36,512
Determine the DNA to be
transcribed

150,618
Catalyze polymerization

155,613
Bind & open DNA template

70,263
Recognize the promoter
for synthesis initiation
•Rifampicin, a bactericidal antibiotic drug, typically
used to treat Mycobacterium infections, including
tuberculosis and leprosy.
•It can bind specifically to the  subunit of RNApol, and inhibit the RNA synthesis.
§1.3 Recognition of Origins
• Promoter
– The promoter is the DNA sequence
that RNA-pol can bind. It is the key
point for the transcription control.
regulatory
sequences
5'
3'
promotor
RNA-pol
structural gene
3'
5'
Prokaryotic promoter
3'
5'
-50
3'
-40
-30
-35
region
TTGACA
AACTGT
Consensus sequence
-20
-10
1
-10
region
10
5'
start
TATAAT
ATATTA
(Pribnow box)
• The -10 region of TATAAT is the region at which a
stable complex of DNA and RNA-pol is formed.
• The -35 region of TTGACA sequence is the
recognition site and the binding site of RNA-pol.
Prokaryotic promoter
TTGACA
TATAAT
ß’


ß

DNA
UP
-35

-10
start
• subunit: recognize the promoter.
•Holoenzyme bind to the promoter.
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: RNA-pol recognizes the
promoter and starts the transcription.
• Elongation: the RNA strand is continuously
growing.
• Termination: 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 (open complex).
• 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 RNA-pol
once the first 3,5 phosphodiester bond
is formed.
• The core enzyme moves along the DNA
template to enter the elongation phase.
The σcycle
• The σfactor dissociates and joins
with a new core polymerase to initiate
another RNA chain.
Transcription initiation complex
RNApol (2) - DNA - pppGpN- OH 3
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
• Transcription bubble: 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.
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.
• Two different strategies for transcription
termination
–  -dependent or  -independent manner.
 -dependent termination
•A protein factor,  factor, destabilizes the interaction
between the template and the mRNA, releasing the
newly synthesized mRNA from the elongation complex.
•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 G-C
rich hairpin loop
followed by a
series of U.
不依赖 -因子的终
止子：含富GC的回文
序列和寡聚U序列。
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 DNADNA hybrid reduces the DNARNA hybrid stability, and
causes the transcription complex
dissociated.
• Among all the base pairings, the
most unstable one is rU:dA.
Transcription
Cycle
Promoter
sigma factor
Terminator
§2.2 Transcription of Eukaryotes
• The eukaryotic RNA-pol requires cofactors to bind to the DNA template
together in the transcription process.
• Eukaryotic systems have three kinds of
RNA polymerases, each of which is a
multiple-subunit protein and responsible
for transcription of different RNAs.
RNA-pol of eukaryotes
RNA-pol
products
Sensitivity to
Amanitin
I
II
III
45S rRNA
hnRNA
5S rRNA
tRNA
snRNA
No
high
moderate
• hnRNA: heterogeneous nuclear RNA
•Amanitin is a specific inhibitor of RNA-pol.
Be careful of
this deadly mushroom!
Amanita phalloides ,or
death cap
α-amanitin
• α-amanitin is a cyclic peptide of eight amino acids. It
is possibly the most deadly of all the amatoxins.
• It is an inhibitor of RNA polymerase II. This
mechanism makes it a deadly toxin.
Transcription of Eukaryotes
a. Initiation
• Transcription initiation needs promoter
and upstream regulatory regions.
• Cis-acting elements: the specific
sequences on the DNA template that
regulate the transcription of one or more
genes.
Cis-acting element
cis-acting element
structural gene
-30
GCGC
CAAT
TATA
exon
intron exon
start
TATA box
enhancer
(Hogness box)
CAAT box
GC box
•Enhancer: DNA sequences that increase the
rate of initiation of transcription by RNA pol II
Transcription factors(TF)
• RNA-pol does not bind the promoter
directly.
• RNA-pol II associates with six
transcription factors, TFII A - TFII H.
• Trans-acting factors: 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
Order of binding is: IID + IIB + RNA poly. II + IIF +IIE +IIH
•TBP: TATA binding protein.
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
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.
• The termination is closely related to the
post-transcriptional modification.
RNA pol can be selectively inhibited
• Rifampicin: a bactericidal antibiotic drug
– It can bind specifically to the  subunit
of the bacterial RNA-pol.
• α-amanitin: a deadly toxin.
– It is an inhibitor of RNA polymerase II.
•Actinomycin D: commonly used
in treatment of a variety of
cancers.
•it can bind DNA duplexes,
and prevent elongation by
RNA polymerase in both
bacteria and eukaryotes.
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
• hnRNA (heteronuclear RNA): primary
transcripts of mRNA.
• 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
5’
m7GpppGp----
G
Capping at the 5- end
• The 5- cap structure is found on hnRNA
too.  The capping process occurs in
nuclei.
• Function:
– stabilize the structure of mRNA
– recognize 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: non-coding region 1~7: coding region
mRNA
Exon and intron
DNA
•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 is
promoted by the
snRNPs (small
nuclear
ribonucleoproteins)
which is a large
RNA-protein
complex:U1,U2,U4,
U5,U6
•Spliceosome:
represent the
snRNP association
with hnRNA at the
exon-intron junction.
lariat
Splicing mechanism
mRNA splicing
• Post-transcriptional modifications of mRNA occur in
the nucleus.
• The mature RNA then enters the cytosol to perform its
function(translation).
d. mRNA editing
• Taking place at
the transcription
level
• One gene
responsible for
more than one
proteins
cytidine deaminase
§3.2 Modification of tRNA
DNA
TGGCNNAGTGC
GGTTCGANNCC
RNA-pol III
tRNA precursor
tRNA processing
• Pre-tRNA requires extensive
processing to become a
functional tRNA.
• Four types of modifications are
involved:
– Removing an extra segment at
the 5' end by RNase P.
– Removing an intron in the
anticodon loop by splicing.
– Replacing two U residues at
the 3'end by CCA.
– Modifying some residues to
characteristic bases.
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 is the precursor of 3 kinds
of rRNAs.
rRNA
18S
28S
5.8S
45S-rRNA
transcription
splicing
18S-rRNA
5.8S and 28S-rRNA
Points
Ⅰ. Prokaryotic transcription
1. RNA polymerase
Sigma (σ) factor +core enzyme = holoenzyme
2. Steps in transcription
a. Initiation: promoter: -10 region(TATAAT), -35
region(TTGACA)
b. Elongation: No primer, no repair, uses NTPs,
transcription bubble
c. Termination:
1). Rho-dependent.
2). Rho-independent.
Points (continued)
Ⅱ. Eukaryotic transcription
1. RNA pol I, II, III.
2. promoter : TATA box, CAAT box, GC box.
3. Transcription factors (TF): TFII
Ⅲ. Post-transcription modification of RNA
1. mRNA: 5’ capping; Addition of poly(A) tail; RNA
splicing, exon, intron
2. rRNA; tRNA.
Ⅳ.Inhibitor
– Rifampicin; α-amanitin; Actinomycin D