Transcription Nuclear

envelope

Nuclear

and
TRANSCRIPTION
DNA envelope

Pre-mRNA
RNA PROCESSING
TRANSCRIPTION
DNA

mRNA
DNA Pre-mRNA
TRANSCRIPTION RNA PROCESSING

mRNA
mRNA

Post
Ribosome TRANSLATION Ribosome DNA
TRANSLATION TRANSCRIPTION

Polypeptide Polypeptide mRNA

Ribosome TRANSLATION Ribosome
TRANSLATION
(a) Bacterial cell (b) Eukaryotic cell
Polypeptide Polypeptide

Transcription
(a) Bacterial cell (b) Eukaryotic cell

Modifications Dr. BONUKE ANYONA

 Learning Objectives
• By the end of this Topic, you should be able
to:
i. Describe the basic principles of transcription
ii. Describe process of transcription
iii. Describe the post transcription modifications
 Basic Principles of Transcription
and Translation
• RNA is the bridge between genes and the
proteins for which they code.
• Transcription is the synthesis of RNA under
the direction of DNA.
• Transcription produces messenger RNA
(mRNA).
• Translation is the synthesis of a polypeptide,
using information in the mRNA.
• Ribosomes are the sites of translation.
 Basic Principles of Transcription
and Translation
• In prokaryotes, translation of mRNA can
begin before transcription is completed.
• In a eukaryotic cell, the nuclear envelope
separates transcription from translation.
• Eukaryotic RNA transcripts are modified
through RNA processing to yield finished
mRNA.
• A primary transcript is the initial RNA
transcript from any gene prior to processing.
 Transcription & Translation
Nuclear
envelope

TRANSCRIPTION
DNA

Pre-mRNA
RNA PROCESSING

mRNA
DNA
TRANSCRIPTION

mRNA
Ribosome TRANSLATION Ribosome
TRANSLATION

Polypeptide Polypeptide

(a) Bacterial cell (b) Eukaryotic cell

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

• The central dogma is the concept that cells

are governed by a cellular chain of
command:
DNA RNA protein
 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

 Replication vs Transcription
• 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
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

5' 3'
3' 5'
 1.2 RNA Polymerase
• The enzyme responsible for RNA synthesis is
DNA-dependent RNA polymerase.
– 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
Determine the DNA to be
 36512
transcribed
 150618 Catalyze polymerization
 155613 Bind & open DNA template
Recognize the promoter
 70263
for synthesis initiation

• 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
products 45S rRNA hnRNA tRNA
snRNA

Sensitivity
No high moderate
to Amanitin

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.
regulatory
Promoter structural gene
sequences
5' 3'
promotor
RNA-pol
3' 5'
 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
 Prokaryotic promoter

• 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.
 Consensus Sequence

Frequency in 45 samples 38 36 29 40 25 30
37 37 28 41 29 44
 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.
 a. Initiation
• The first nucleotide on RNA transcript is
always purine triphosphate. GTP is more
often stable 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

 a. Initiation

• 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.
 b. Elongation
• The release of  subunit causes
conformational change of core enzyme. The
core enzyme slides on DNA template toward
the 3 end.
• Free NTPs are added sequentially to the 3 -
OH of the nascent RNA strand.
 b. Elongation
• RNA-pol, DNA segment of ~40nt and the
nascent RNA form a complex called the
transcription bubble.
• The 3 segment of nascent RNA hybridizes
with the DNA template, and its 5 end extends
out the transcription bubble as the synthesis is
processing.
Transcription bubble
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.
 rplL protein
DNA
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 RNA-pol moving.
• Then the competition of RNA-RNA hybrid
and the DNA-DNA hybrid reduces the DNA-
RNA hybrid stability, and causes
transcription complex to 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 specific
sequences on DNA template that regulate
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)

enhancer CAAT box

GC box
 Transcription factors

• RNA-pol does not bind the promoter

directly.
• RNA-pol II associates with six
transcription factors, TFII A - TFII H.
• Trans-acting factors are proteins that
recognize and bind directly or indirectly to
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
 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.
 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
promoter recognition
The formation of the active eukaryotic
initiation complex.
The diagrams represent the complexes
formed on the TATA box by the
transcription factors and RNA polymerase
II. (A) The TFIID complex binds to the
TATA box through its TBP subunit.
(B) TFIID is stabilized by TFIIA. (C)
TFIIB and TFIIH join the complex on the
TATA box while TFIIE and TFIIF
associate with RNA polymerase II. (D)
RNA polymerase is positioned by TFIIB,
and carboxy-terminal
its
domain (CTD) is bound by TFIID.
(E) The CTD is phosphorylated by TFIIH
and is released by TFIID. The RNA
polymerase II is now competent to
transcribe mRNA from the gene.
 Promoter recognition: enhancers

• Eukaryotic genes may also have enhancers.

• Enhancers are DNA sequences that can
control efficiency and rate of transcription.
• They do not have to be close to the gene
promoter that they regulate, and must be
located on the same chromosome.
Promoter recognition: enhancers
• Enhancers regulate the expression of a gene
in specific cell types, and they control the
timing of gene expression.
• Enhancers are critical for normal
development because they regulate the timing
and tissue-specificity of gene expression.
 Enhancer locations
• Enhancers can be located at great distances
from the gene they regulate, either 5´ or 3´ of
the transcription start, in introns or even on
the noncoding strand.
• One of the most common ways to identify
promoters and enhancers is to use a reporter
gene.
• Reporter genes are artificial gene expression
systems that can be used to identify regions in
DNA that are important for regulating gene
expression.
 Promoter recognition:
enhancers

• Modular transcriptional regulatory regions using Pax6 as an

activator. (A) Promoter and enhancer of the chick lens δ1 crystallin
gene. Pax6 interacts with Sox2 and Maf to activate this gene. (B)
Enhancer of the rat somatostatin gene. Pax6 activates this gene by
cooperating with the Pdx1 transcription factor.
 b. Elongation
• RNA polymerase moves along the transcribed or
template DNA strand.
• The new RNA molecule (primary transcript) forms a
short RNA-DNA hybrid molecule with the DNA
template.
• 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
post-
transcriptional
modification.
 Section 3

Post-Transcriptional
Modification
3.1 Nascent RNA
• 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

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
• 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.
 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
O OH
Pi
O P O AAAAA-OH 3'
O
 b. Poly-A tailing at 3- end
• There is no poly(dT) sequence on the DNA
template.
 The tailing process does 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 2 3 4 5 6 7
A B C D E F 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 non-coding sequences that are

transcribed into primary mRNAs, and will be
cleaved out in the later splicing process.
mRNA splicing
Splicing mechanism
lariat
 d. mRNA editing

• Takes place at the transcription level

• One gene responsible for more than one
proteins
• Significance: gene sequences, after post-
transcriptional modification, can undergo
multiple purpose differentiation.
 Different pathway of apo B

Human apo B gene

hnRNA (14,500 base)

CAA to UAA
At 6666
Liver
apo B100
（ 50 kD ） Intestine
apo B48
（ 24 kD ）
3.2 Modification of tRNA
Cleavage

RNAase P
endonuclease

ligase
Addition of CCA-OH

tRNA nucleotidyl
transferase

ATP ADP
 Base modification

1. Methylation
（2） （1） A→mA, G→mG
（1）
2. Reduction
U→DHU
3. Transversion U→ψ
4. Deamination
（3） A→I
（4）
3.3 Modification of rRNA
• 45S transcript in nucleus is the precursor
of 3 kinds of rRNAs.

• Matured rRNA will be assembled with

ribosomal proteins to form ribosomes
that are exported to cytosolic space.
3.3 Modification of rRNA

rRNA

18S 5.8S 28S 45S-rRNA

transcription

splicing
18S-rRNA
5.8S and 28S-rRNA
 3.4 Ribozyme

• The catalytic RNA is called ribozyme.

• Self-splicing happens often for intron I and
intron II.
• Both the catalytic domain and the substrate
are located 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.