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, G-C

Processing No Yes
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.
Sense and antisense strand
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
strand
3' CGTCATGTACAG 5' 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.
 Asymmetric transcription
• Each of the two DNA strands of a given
gene serves as an RNA template, each gene
would produce two RNA products with
complementary sequences, which should
code for two different proteins.

• Asymmetric transcription produce mRNA

of different size, molecular content, or
developmental potential.
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   ω .

holoenzyme 
core enzyme  


 RNA-pol of E. Coli

subunit MW function
Determine the DNA to be
 36512
transcribed

 150618 Catalyze polymerization

 155613 Bind & open DNA template

Recognize the promoter
 70263
for synthesis initiation
ω: smallest subunit and facilitates assembly of RNAP and
stabilizes assembled RNAP
• Rifampicin, a therapeutic drug for
tuberculosis treatment, can bind specifically
to the  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 of eukaryotes

RNA-pol I II III
5S rRNA
hnRNA
Products 45S rRNA tRNA
snRNA
Recognition of Origins
• Each transcriptable region is called operon.
• One operon includes several structural genes and
upstream regulatory sequences (or regulatory
regions).
 Promoter

regulatory
structural gene
sequences
5' 3'
promotor
RNA-pol
3' 5'

• The promoter is the DNA sequence that RNA-

pol can bind.
• It is the key point for the transcription control.
 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
• 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.
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.
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.
Transcription initiation complex:
RNA-pol (2) - DNA – new RNA nucleotide

Closed complex

The initial binding between

the polymerase and a
promoter, because the DNA is
not unwound
• No primer is needed for RNA synthesis.
• The  subunit falls off from the RNA-pol
after addition of about the first 10 nucleotides.
• 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 (NMP)n+1 + PPi

elongated
RNA strand substrate 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
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.

Rho bind to a specific site on the RNA and pulls the RNA off
the RNA polymerase.
 -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.
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)
-25bp
enhancer CAAT box
-70bp
GC 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 E
TF II TBP TAF
TF II
A TATA B
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 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.
c. Termination

• The termination sequence is AATAAA

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

The matured mRNAs are much shorter than the DNA

templates.
 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.
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.