DNA Transcription in Prokaryotic and Eukaryotic Cell , Post Transcription modification, Reverse Transcription.

1. DNA TRANSCRIPTION
Rikesh Lal Shrestha
M.Pharm (2015), Industrial
Kathmandu University

2. Contents
• INTRODUCTION
• DNA TRANSCRIPTION STAGES
• POST TRANSCRIPTION MODIFICATION
5’ CAPPING
POLY (A) TAIL
SPLICING
• REVERSE TRANSCRIPTION
• DRUGS INHIBITING TRANSCRIPTION

3. Introduction
• The synthesis of an RNA molecule from DNA molecule in the
presence of enzyme RNA polymerase is called Transcription.
• Information stored in DNA is transferred to RNA molecules
during transcription.
• All eukaryotic cells have five major classes of RNA:
ribosomal RNA (rRNA), messenger RNA (mRNA), transfer
RNA (tRNA), small nuclear RNA and microRNA (snRNA and
miRNA).
• The first three are involved in protein synthesis, while the
small RNAs are involved in mRNA splicing and regulation of
gene expression.

4. Similarities between Replication and
Transcription
The processes of DNA and RNA
synthesis are similar in that
they involve-
(1) The general steps of
initiation, elongation, and
termination with 5' to 3'
polarity;
(2) Large, multicomponent
initiation complexes; and
(3) Adherence to Watson-Crick
base-pairing rules.

5. Replication Transcription
DNA replication is catalyzed by DNA
polymerase which needs a primer.
RNA synthesis is catalyzed by RNA
polymerase
Deoxyribonucleotides are used in DNA
synthesis
Ribonucleotides are used in RNA
synthesis
Thiamine is the complementary base pair
for Adenine in DNA.
Uracil is the complementary base pair for
Adenine in RNA.
The entire genome must be copied during
DNA replication
Only a portion of the genome is
transcribed or copied into RNA
There is proofreading function during
DNA replication.
There is no proofreading function during
RNA transcription.
Differences between Replication and Transcription

6. Template strand
•The DNA strand that is transcribed or
copied into an RNA molecule is called
template strand.
•The another non-template DNA strand,
is called coding strand.
•The information in the template strand is
read out in the 3' to 5' direction
•The information in the RNA molecule is
read out in the 5' to 3' direction
•In the coding strand the sequence is
same as that of the sequence of RNA
primary transcript. With the exception of
T for U changes.

7. Transcription unit
• A transcription unit is defined as that region of DNA that includes the signals for
transcription initiation, elongation, and termination.
• The nucleotide in the promoter adjacent to the transcription initiation site is designated -1,
• These negative numbers increase as the sequence proceeds upstream, away from the
initiation site
• The nucleotide in the initiation site is designated +1,
• These positive numbers increase as the sequence proceeds downstream, away from the
initiation site

8. Bacterial DNA-Dependent RNA Polymerase
The DNA-dependent RNA
polymerase (RNAP) of the
bacterium Escherichia coli exists as
an approximately 400 kDa core
complex consisting of-
•two identical α subunits,
•similar but not identical β and β '
subunits, and
•an ω subunit and a
•A sigma subunit (σ)
•Beta is thought to be the catalytic
subunit.

9. Bacterial DNA-Dependent RNA Polymerase
• RNAP, a metalloenzyme, also contains two zinc
molecules.
• The core RNA polymerase associates with a
specific protein factor (the sigma σ factor) that
helps the core enzyme recognize and bind to the
specific deoxynucleotide sequence of the
promoter region to form the preinitiation
complex (PIC)
• Bacteria contain multiple factors, each of which
acts as a regulatory protein.

10. Mammalian DNA-Dependent RNA
Polymerases
Mammalian cells possess three distinct nuclear
DNA-Dependent RNA Polymerases
• RNA polymerase I is for the synthesis of
rRNA
• RNA polymerase II is for the synthesis of
mRNA and miRNA
• RNA polymerase III is for the synthesis of
tRNA/5S rRNA, snRNA

11. Prokaryotic versus Eukaryotic Transcription
Parameters Prokaryotic Cell Eukaryotic Cell
Location Transcription Occur in
cytoplasm of Prokaryotic cell
Transcription Occur in nucleus of
Eukaryotic cell
RNA
Polymerase
Single RNA polymerase have
capability to produce rRNA,
mRNA & tRNA.
Three classes of RNA polymerases (I,
II,III). RNA Poly. I rRNA, RNA Poly.II
mRNA, RNA Poly.III tRNA
Synthesized
RNA
The RNA produce is fully
functional for translation
process.
The RNA produced is not fully functional
and k/a premature m-RNA which need to
be modified to produce mature m-RNA
that can synthesize protein.
Process Transcription along with
translation process take place.
So the process is also k/a
transcription coupled
translation.
After transcription complete then only
translation process take place.
Transcribed
RNA
Transcribed RNA is
polycistronic (i.e multiple
genes are regulated by only
one promoter) . So, multiple
proteins are produce in chain.
Transcribed RNA is monocistronic (i.e
single gene is placed on single promoter)

12. Stages of Transcription
Both prokaryote and eukaryote have 3 stage
of transcription. They are:
• Initiation
• Elongation
• Termination

13. Initiation in Prokaryote
• The RNA polymerase and sigma factor combine to form a molecule
called holoenzyme.
• This holoenzyme binds to the promoter region of double strands DNA
forming closed complex.
• Then holoenzyme unwind DNA helix , thus open promoter complex is
formed.
• After 10–20 nucleotides have been polymerized, holoenzyme undergoes
conformational change leading to promoter clearance.
• Once this transition occurs, holoenzyme moves away from the promoter,
transcribing down the transcription unit, leading to the next phase of the
process, elongation.

14. Elongation in Prokaryote
• As the elongation complex that is holoenzyme progresses along the DNA
strands, it unwinding DNA to provide access for the complementary base
pairing to the nucleotides of the template strand.
• After addition of few riboneucleotides the sigma factor dissociate from
holoenzyme . Then RNA polymerase adds nucleotides to 3’ –OH group.
Thus elongation proceed in the direction of core enzyme.

15. Termination in Prokaryote
• Two types of terminator sequences
occur in prokaryotes:
•Rho() Independent: In RNA sequence
there occurs G and C rich region, their
Palindromic repeat forms a hairpin loop.
Beyond the hair pin, RNA sequence
contains a strings of Us, the bonding of
Us to the corresponding As is weak. This
facilitates the dissociation of the RNA
from DNA.
•Rho() dependent: A sequence of DNA
template strand signal is recognized by
protein k/a Rho () factor proteins, it
attach and climb RNA strand then break
the hydrogen bonds between the
template DNA and RNA .

16. Initiation in Eukaryote
We can divide eukaryotes promoter into two regions:
The core promoters elements. The best characterized are TATA Box = TATAAAA,
located at about position -30 bp(*AT-rich DNA is easier to denature than GC-rich
DNA)
Promoter proximal elements (located upstream, ~-50 to -200 bp) “Cat Box” =
CAAT and “GC Box” GGGCGG
 General Transcription factors (GTF) are proteins that help eukaryotic RNA
polymerase recognize promoter sequences.
 Binding of GTFs and RNA polymerase occurs in set of order.
 Complete complex (RNA polymerase + GTFs) is called a pre-initiation complex
(PIC).
 Transcription regulatory proteins (activators) bind to the enhancers region in
ds DNA and also to RNA polymerase II in order to increase the rate of
transcription initiation of eukaryotic genes.

17. Initiation in Eukaryote
Order of binding is: IID + IIA + IIB + RNA poly. II + IIF +IIE +IIH

18. Elongation in Eukaryote
• As RNA polymerase moves along the DNA it continues to
untwist the double helix, exposing about 10 to 20 DNA bases
at a time for pairing with RNA nucleotides.
• RNA polymerase synthesizes a single strand of RNA against
the DNA template strand (anti-sense strand), adding
nucleotides to the 3’ end of the RNA chain.
Elongation
RNA
polymerase
Non-template
strand of DNA
RNA nucleotides
3 end
A E G C A
U
T A G G T T
A
T C C A A
3
5
5
Newly made
RNA
Direction of transcription
(“downstream”) Template
strand of DNA

19. Termination in Eukaryote
• Specific sequences in the DNA signal termination of
transcription (AAUAAA)
• When one of these is encountered by the polymerase, the
RNA transcript is released from the DNA and the double
helix can zip up again.

20. POST TRANSCRIPTION MODIFICATION
5’ capping
• It is a 7-methylguanosine tri-phosphate cap structure at the
5' terminal of eukaryotic mRNA.
• The cap structure is added to the 5' end of the newly
transcribed mRNA before it transport to cytoplasm.
• The 5' cap of the RNA transcript is required both for efficient
translation initiation and protection of the 5' end of mRNA
from attack by 5-'3' exonucleases.

21. 5’ capping
•There is addition of the Guanosine triphosphate to 5’terminal of mRNA
by enzyme guanylyl transferase.
•The 5’-end of the mRNA is capped 5’ to 5’ with a guanine nucleotide
forming triphosphate bridge.
•Methylation occurs in N7 of guanine by guanine-7-methyl transferase.
•Additional methylation steps may occur.
•The secondary methylations of mRNA molecules, those on the 2'-
hydroxy and the N6 of base residues, occur after the mRNA molecule has
appeared in the cytoplasm.

22. Poly (A) tail
• Poly(A) tails are added to the 3' end of mRNA molecules in a posttranscriptional
processing step.
• The mRNA is first cleaved about 20 nucleotides downstream from an AAUAAA
recognition sequence.
• Another enzyme, poly(A) polymerase adds a poly(A) tail which is subsequently
extended to as many as 200 A residues.
• The poly(A) tail appears to protect the 3' end of mRNA from 3' -5' exonuclease
attack.

23. RNA Splicing
• Splicing : Removal of introns
• Introns: non-coding sequences
between exons
• Exons: amino acid coding sequences
are spliced.
• “snurps” snRNP bind to splice site and
form a spliceosome
• Spliceosome excises the intron and
rejoins the exons.
• Mature mRNA is produce that moves
into cytosol from nuclear pore for
protein synthesis.

24. Reverse Transcription
• It is the process of synthesis double stranded
DNA from Single stranded RNA by reverse
transcriptase enzyme (RNA directed DNA
polymerase).
• Reverse transcriptase common in HIV,
MMLV(Moloney Murine Leukemia Virus),
AMV(Avian Myeloblastosis Virus)
• Reverse transcriptase enzyme includes two
activity: DNA polymerase and RNAase H

25. Retrovirus Cycle

26. Drug Inhibiting Transcription
• Rifampicin binds with Beta subunit of
prokaryotic RNA polymerase but not to
eukaryotic RNA polymerases. Rifampicin use
for the treatment of tuberculosis and leprosy.
• Mitomycin used as anticancer drug
Intercalates with DNA strands blocks
transcription.
• Alpha amanitin is a molecule made from the
“death cap” mushroom and is a known potent
inhibitor RNA polymerase. The mechanism of
action is that alpha amanitin inhibits RNA
polymerase –II at both the initiation and
elongation states of transcription.
• Actinomycin D- Intercalates with DNA
strands .Actinomycins inhibit both DNA
synthesis and RNA synthesis by blocking
chain elongation. Actinomycins are used as
anticancer drugs

27. REFERENCE
• Professor (Dr.) Namrata Chhabra Biochemistry For Medics-
Lecture Notes
• Chapter 12 of Molecular Biology of the Gene 6th Edition
(2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine,
M, Losick, R. 377-414.
• Murakami KS, Darst SA. (2003) Bacterial RNA polymerases:
the wholo story. Curr Opin Struct Biol 13:31-9.
• Campbell, E, Westblade, L, Darst, S., (2008) Regulation of
bacterial RNA polymerase factor activity: a structural
perspective. Current Opinion in Micro. 11:121-127
• John Wiley & Sons, Inc, Transcription and RNA Processing
• Biochemistry For Medics- Lecture Notes.

28. THANK YOU……!!!!