DNA transcription

Stryer 6th/8th edition, Chapter 4, 29

 Contents
• Prokaryotic transcription
– Promoter
– E. coli RNA polymerase and Sigma factor
– Process of transcription: initiation, elongation and
termination
– mRNA, tRNA, rRNA and processing
• Antibiotics that inhibit transcription
• Eukaryotic transcription
– RNA polymerase, common element at promoter, enhancer
– Posttranscriptional modification: CAP, tail, RNA editing
Promoter啟動子 sites

• Sequences of DNA that direct the RNA polymerase to the proper

initiation site for transcription

• Consensus sequences deduced from analyses of many promoters. The

first nucleotide (the start site) of a transcribed DNA sequence is denoted
as +1 and the second one as +2; the nucleotide preceding the start site
is denoted as -1.

• Two sequence motifs are identified in prokaryotic promoters:

-10 sequence and -35 sequence, separated by 17 nucleotides at
optimal

3
Strength of promoters

• The strength of a promoter sequence serves to regulate

transcription

• Genes with strong promoters are transcribed frequently, once in every 2

seconds in E. coli

• Genes with very weak promoters are transcribed about once in 10 mins

• E.coli has about 2000 promoter sites

4
E. coli RNA polymerase

• ~ 400 kDa enzyme consisting of four kinds of subunits

• Core enzyme: α2ββ’ (contains catalytic site, a small subunit ω is
also included recently)
• Holoenzyme : α2ββ’σ
 role of σ subunit - recognition of promoter site
- once transcription is initiated, dissociates
from the rest of the enzyme
• In E. coli, one single RNA polymerase transcribes all types of RNA.

5
Active site of RNA polymerase

• two metal ions : one bound to the

enzyme, whereas the other appears to
come in with the nucleoside
triphosphate and leave with the
pyrophosphate.

• three conserved aspartate residues of

the enzyme participate in binding these
metal ions.

6
Sigma Factor of RNA polymerase

 decreases the affinity of RNA polymerase for general regions of DNA by a 104
fold. In its absence, the core enzyme binds DNA tightly.
 The holoenzyme binds to duplex DNA and moves along the double helix in
search of a promoter.
 The σ subunit is released when the nascent RNA chain reaches nine or ten
nucleotides in length

Structure of the σ subunit. The structure

of a fragment from the E.coli subunit σ70
reveals the position of an α-helix which plays
an important role in binding to the
-10 TATAAT sequence. 7
Alternative Promoter Sequences

• E. coli contains multiple σ factors to recognize several types of

promoter sequences
σ70 : recognizes the consensus sequences.
σ32 : recognizes the promoters of heat-shock genes. The increased
transcription of heat-shock genes leads to the coordinated
synthesis of a series of protective proteins.
Other σ factors: respond to environmental conditions, such as
nitrogen starvation.
8
Initiation of Transcription

• Duplex DNA must be unwound before RNA synthesis

• Each bound polymerase molecule unwinds a 17bp segment of DNA

• The RNA chains, like DNA chains, grow in the 5’ → 3’ direction

9
Overview of the transcription initiation
• In binding phase, interaction of RNA polymerase
with promoter leads to the formation of a closed
complex – promoter is stably bound but not
unwound
• In initiation phase, a 12-15 bp region of DNA
within the -10 region to +2 or +3 is unwound to
form an open complex
• Once elongation commences, σ subunit is released
and polymerase leaves the promoter for the
elongation

10
Fig. 26-6 Leh
Elongation of transcription
• The newly synthesized RNA forms a RNA-DNA helix (about 8 bp long, which
corresponds to nearly one turn of a double helix)

• About 17 bp of DNA are unwound throughout the elongation phase

• Rate of elongation is about 50 nucleotides per second

A transcription bubble in the elongation of an RNA transcript. Duplex DNA is

11
unwound at the forward end of RNA polymerase and rewound at its rear end.
Elongation of Transcription

The length of the RNA-DNA hybrid is

determined by a structure within the RNA
polymerase that forces the RNA-DNA
hybrid to separate, allowing the RNA chain
to exit from the enzyme and the DNA chain
to rejoin its DNA partner.

12
Fidelity of Transcription

• Proofreading: RNA-DNA hybrid can move in the direction opposite

that of elongation for hydrolysis reaction

• The error rate of RNA synthesis is of the order of one mistake per
104 or 105 nucleotides, about 105 times as high as that of DNA
synthesis.

• Question: Why higher error rate in transcription can be tolerated?

13
Termination of Transcription
1. Protein-independent Termination of Transcription

The transcribed regions of DNA templates contain stop signals

e.g. a palindromic GC-rich region followed by an AT-rich region
 RNA transcript is self-complementary
 form a hairpin structure with a stem and loop, a structure
favoured by its high content of G and C residues. This stable
hairpin is followed by a sequence of four or more uracil residues
 RNA polymerase pauses after RNA folds into a hairpin. DNA-
14
RNA hybrid produced after the hairpin is unstable
Protein-independent Termination of Transcription

• RNA polymerase pauses immediately after it has synthesized a

stretch of RNA that folds into a hairpin.

• the RNA-DNA hybrid helix produced after the hairpin is unstable

because its rU-dA base pairs are the weakest. Hence, the pause
in transcription caused by the hairpin permits the weakly bound
nascent RNA to dissociate from the DNA template and then from
the polymerase.

• the solitary DNA template strand rejoins its partner to re-form the
DNA duplex, and the transcription bubble closes.

15
2. Protein-dependent Termination of Transcription

The termination of transcription can be protein-dependent that requires

the participation of an additional factor, the rho (ρ) factor. Hexameric ρ
specifically binds single-stranded RNA.
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2. Protein-dependent Termination of Transcription

• Hexameric ρ specifically binds single-stranded RNA. It prefers DNA with rich C but
poor G.

• It migrates at 5’ to 3’ direction until it reaches the transcription complex.

• When ρ catches RNA polymerase at the transcription bubble, it breaks the RNA-DNA
hybrid helix by functioning as an RNA-DNA helicase. It pulls the RNA away from RNA
17
polymerase and DNA template.
What is the fate of the RNA transcript after transcription?

• Messenger RNA (信使核糖核酸 mRNA)

• Transfer RNA (轉運核糖核酸 tRNA)
• Ribosomal RNA (核糖體核糖核酸 rRNA)

Messenger RNA (mRNA)

 RNA transcript required as a template for synthesis of polypeptide
chain
 contains ribosome binding site, translation initiation site and
coding sequence and translation termination site
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Cellular mRNA are degraded at different rates

Concentration of cellular mRNA depends on :

1. Rate of synthesis
2. Rate of degradation

Degradation of mRNA ensure that mRNAs do not build up in the cell and direct
the synthesis of unnecessary proteins

• Half-life of bacterial mRNA ~ 1.5 min

• mRNA is degraded by ribonucleases:-
- one or a few cuts by endoribonuclease
- followed by 3’  5’ degradation by exoribonucleases
• Hairpin structure in bacterial mRNAs with a ρ-independent terminator confers
stability against degradation. A full length mRNA with hairpin structure at the
3’ end is more stable. 19
Transfer RNA (tRNA)

 65 -110 nucleotides long with modified

nucleotides

 Cloverleaf secondary structure

 Modified nucleotides (e.g. methylated

guanine and dihydrouracil) affect tRNA
structure and stability

 CCA at 3’ end – acceptor site of amino

acid (each tRNA has an attachment site
for a specific amino acid)

 Anticodon反密碼子: three-base
nucleotide sequence that recognizes a
specific codon on the mRNA
20
 Ribosomal RNA (rRNA)

 extensive regions of secondary structure

arising from base pairing of complementary
sequences

 it is the catalytic component of ribosome,

crystal structure of large subunit of bacterial
ribosome shows that the active site for
peptide bond synthesis is composed of 23S
rRNA only.

Ribosome structure. The structure of a part of the

ribosome showing the site at which peptide bond
formation takes place. This site contains only RNA 21
(yellow), with no protein (red).
Processing of the Precursors of Transfer (tRNA)
and Ribosomal RNA (rRNA)

In prokaryotes, messenger RNA molecules undergo little

or no modification after synthesis by RNA polymerase.
Indeed, many mRNA molecules are translated while
they are being transcribed.

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Processing of the Precursors of tRNA and rRNA

• Transfer RNA and ribosomal RNA molecules are generated by cleavage and
other modifications of nascent RNA chains. Specific nucleases are required to
cleave these precursors of rRNA and tRNA. Cleavage is precise.

• E.g. in E. coli, three kinds of rRNA molecules and a tRNA molecule are excised
from a single primary RNA transcript that also contains spacer regions.

• Other transcripts contain arrays of several kinds of tRNA or of several copies of

the same tRNA.
23
Processing of the Precursors of tRNA and rRNA

A second type of processing is the addition of nucleotides to the

termini of some RNA chains.

 e.g. CCA, a terminal sequence required

for the function of all tRNAs, is added to
the 3’ ends of tRNA molecules that do not
already possess this terminal sequence.

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Processing of the Precursors of tRNA and rRNA

A third type of processing is the modification of bases and ribose units

of ribosomal RNAs.

• some bases of rRNA are methylated

• unusual bases are found in all tRNA

molecules. They are formed by the enzymatic
modification of a standard ribonucleotide in a
tRNA precursor.

• E.g. uridylate residues are modified after

transcription to form ribothymidylate and
pseudouridylate. These modifications generate
diversity, allowing greater structural and
functional versatility.
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Transcription and translation processes are
closely coupled in prokaryotes

The translation of bacterial mRNA begins while the transcript is still

being synthesized. The separation of transcription and translation
enables eukaryotes to regulate gene expression in much more intricate
ways, contributing to the richness of eukaryotic form and function. 26
Antibiotics Inhibitors of Transcription

1. Rifampicin利福平

Rifampicin is a semisynthetic derivative of rifamycins, which are

derived from a strain of Streptomyces鏈黴菌屬. This antibiotic
specifically inhibits the initiation of RNA synthesis.
27
Antibiotics Inhibitors of Transcription

• Rifampicin interferes with the formation of the first few phosphodiester bonds in the RNA.

• The structure of a complex between a prokaryotic RNA polymerase and rifampicin reveals
that the antibiotic binds to a pocket that is normally occupied by the newly formed DNA–
RNA hybrid.

• It blocks the channel into which the RNA-DNA hybrid generated by the enzyme must pass.

• Rifampicin does not hinder chain elongation once initiated, because the RNA-DNA hybrid
present in the enzyme prevents the antibiotic from binding. 28
Antibiotics Inhibitors of Transcription

2. Actinomycin D 放線菌素

• a polypeptide-containing antibiotic from a

different strain of Streptomyces

• binds tightly and specifically to double-helical

DNA and prevents it from being an effective
template for RNA synthesis.

Actinomycin D is extensively used as a highly specific inhibitor of the

formation of new RNA in both prokaryotic and eukaryotic cells. Its ability
to inhibit the growth of rapidly dividing cells makes it an effective
therapeutic agent in the treatment of some cancers.
29
Which of the following is true of RNA
synthesis (transcription)?
A. RNA synthesis is always in the 5' - 3'
direction.
B. RNA polymerase needs a primer to
initiate transcription.
C. In transcription, U is inserted opposite T.
D. New nucleotides are added on to the 2'
OH of the ribose sugar.

30
 Eukaryotic RNA polymerases

• RNA polymerase I makes pre-ribosomal RNA, contains the precursor of

18S, 5.8S and 28S rRNAs, which is processed to form the mature rRNA
• RNA polymerase II makes mainly mRNA
• RNA polymerase III makes tRNAs and other small RNA, some sequences
required for the regulated initiation of transcription are located with the
gene itself
• Organelle specific RNA polymerases: in mitochondria and chloroplasts
31
 Eukaryotic RNA polymerases
• Eukaryotic RNA polymerase II is a large protein, containing from 8
to 14 subunits (12 subunits in yeast) and having total molecular
masses greater than 500 kD.
• RNA polymerase II contains a unique carboxyl-terminal domain
on the 220-kD subunit called the CTD.
• In yeast CTD, there are 27 repeats of –YSPTSPS-.
• CTD is separated from the main body by an unstructured linker
sequence
• The activity of RNA polymerase II is regulated by phosphorylation
mainly on the serine residues of the CTD.

32
 In eukaryotes, common elements can be found
in the RNA polymerase II promoter region

TATA box
The TATA box is usually
found between positions -30
and -100.
⇒ Resembles the prokaryotic -10
sequence (TATAAT), but farther
from the start site.

• The mutation of a single base in the TATA box

markedly impairs promoter activity.
⇒ The precise sequence, not just a high content of AT pairs,
is essential. 33
 In eukaryotes, common elements can be found
in the RNA polymerase II promoter region

Initiator element (Inr)

• The TATA box is often
paired with an Initiator
element (Inr), a sequence
found at the transcriptional
start site, between
positions -3 and + 5.

• It defines the start site because the other promoter

elements are at variable distances from that site.
⇒ Its presence increases transcriptional activity. 34
In eukaryotes, common elements can be found
in the RNA polymerase II promoter region

DPE
– The downstream core promoter element (DPE)
is commonly found in conjunction with the Inr
in transcripts that lack the TATA box.
– In contrast with the TATA box, the DPE is
found downstream of the start site, between
positions +28 and +32.

35
In eukaryotes, common elements can be found
in the RNA polymerase II promoter region

Additional regulatory sequences

• Many promoters contain a CAAT box,
and some contain a GC box.
• Located between -40 and -150.

 Constitutive genes (genes that are continuously expressed

rather than regulated) tend to have GC boxes in their
promoters. 36
In eukaryotes, common elements can be found
in the RNA polymerase II promoter region

Enhancer sequence增强子
 Enhancer sequences can stimulate transcription
at start sites thousands of bases away.
 Enhancers increase expression of a gene many-fold.
 They can be upstream, downstream, or even in
the midst of a transcribed gene.

37
 Transcription at
Assembly of RNA polymerase and
transcription factors at promoter to
RNA polymerase
form basal transcription apparatus
II promoters

After termination,
Pol II is released,
dephosphorylated,
and recycled.

Elongation is accompanied by the

release of many transcription
factors and is also enhanced by
An overview of transcription:
elongation factors (e.g. elongin). https://www.youtube.com/watch?v=WsofH466lqk
The carboxyl-terminal domain
of the largest Pol II subunit is 38
phosphorylated by TFIIH
 Posttranscriptional modification of
mRNA - Addition of 5’ CAP
• 5’ triphosphate of nascent RNA is
immediately modified
• A phosphoryl group is hydrolyzed.
The diphosphate 5’ end then attacks
the α-phosphorus atom of GTP to
form 5’-5’ triphosphate linkage, which
is called a CAP
• N-7 of terminal guanine is then
methylated to form cap 0. Adjacent
riboses may be methylated to form
cap 1 or cap 2.
• Caps protect 5’ end of mRNA from
phosphatases and nucleases
• Caps for binding of translation
initiation factor, for enhancing
translation rate
39
Posttranscriptional modification of mRNA - Addition
of tail
• An endonuclease tethered to the carboxyl-terminal domain of
the largest subunit of PolII recognizes the sequence AAUAAA
and cleaves about 20 nucleotides downstream
• Poly(A) polymerase adds about 250 A to the 3’ end of the
transcript, ATP is needed
• For stabilization of RNA
• Rate of translation ∝ length of poly A

Comparison between
prokaryotic and eukaryotic
transcription:
https://www.youtube.com/
watch?v=b60upy8BFH4

40
Which of the following occur in both
eukaryotic and bacterial transcription?
1. 5' cap.
2. polyA tail.
3. Promoter.
4. DNA-dependent RNA polymerase.
A. 1, 2
B. 1, 3
C. 2, 4
D. 3, 4
41
 Posttranscriptional modification of
mRNA - RNA editing核糖核酸編輯
• Change of nucleotide sequence after
RNA is made
• e.g. Apolipoprotein B載脂蛋白– two
forms
• Apo B-100, synthesized in liver, for
transport of lipids synthesized in the
cell
• Apo B-48, synthesized in small
intestine, carries dietary fat in the form
of chylomicrons. This form cannot
bind to low-density-lipoprotein
receptor on cell surfaces
• Apo B-48 is generated by deaminating
a specific cytidine residue so that CAA
(Gln) is changed to UAA (stop)
• This reaction is present in small 42
intestine
 Posttranscriptional modification of
mRNA - RNA editing
• The reaction requires a multi-
component enzyme apobec-1 and
transcription factors
• The regulation depends on
developmental, hormonal and
nutritional stages which regulate the
expression of apobec-1 mRNA

Cytosine Uracil
43