Transcription in eukaryotes: A brief view Transcription is the process by which single stranded RNA is synthesized by double stranded DNA. Transcription in eukaryotes and prokaryotes has many similarities while at the same time both showing their individual characteristics due to the differences in organization. RNA Polymerase (RNAP or RNA Pol) is different in prokaryotes and eukaryotes. Coupled transcription is seen in prokaryotes but not in Eukaryotes. In eukaryotes the pre-RNA should be spliced first to be translated. In Eukaryotic transcription, synthesis of RNA occurs in the 3’→5’ direction. The 3’ end is more reactive due to the hydroxide group. 5’ end containing phosphate groups meanwhile, is not very reactive when it comes to adding new nucleotides. In Eukaryotes, the whole genome is not transcribed at once. Only a part of the genome is transcribed which also acts as the first, principle stage of genetic regulation. Eukaryotes have five nuclear polymerases: • RNA Polymerase I: This produces rRNA (23S, 5.8S, and 18S) which are the major components in a ribosome. This also produces pre-rRNA in yeasts. • RNA Polymerase II: Helps in the production of mRNA (messenger RNA), snRNA (small, nuclear RNA), miRNA. This is the most studied type and requires several transcription factors for its binding • RNA Polymerase III: This synthesizes tRNA (transfer RNA), 5S rRNA and other small RNAs required in the cytosol and nucleus. • RNA Polymerase IV: Synthesizes siRNA (small interfering RNA) in plants. • RNA Polymerase V: This is the least studied polymerase and synthesizes siRNA-directed heterochromatin in plants. Eukaryotic transcription can be broadly divided into 4 stages: • Pre-Initiation • Initiation • Elongation • Termination Transcription is an elaborate process which cells use to copy the genetic information stored in DNA into RNA. This pre-RNA is modified into mRNA before being transcribed to proteins. Transcription is the first step to utilizing the genetic information in a cell. Both Eukaryotes and Prokaryotes employ this process with the basic phases remaining the same. However eukaryotic transcription is more complex indicating the changes transcription has undergone towards perfection during evolution.

1. Tanvi Potluri

2. Eukaryotic
Transcription

3. Overview
• Introduction
• Transcription in Prokaryotes and Eukaryotes
• Pre-Initiation
• Initiation
• Elongation
• Termination
• Post- Transcription

4. Transcription
DNA → RNA
4 Stages:
1. Pre- Initiation
2. Initiation
3. Elongation
4. Termination

5. Introduction
Synthesis of single stranded RNA
5’→ 3’ direction
RNA Polymerase is used
No primers needed
Only a part of the genome is transcribed
First stage of gene expression and the principle
conservation step.

6. RNA Polymerase
• 3 Nuclear Polymerases
1. RNA Pol I- Produces rRNA (28S, 5.8S, 18S)
2. RNA Pol II- Produces mRNA, snRNA, siRNA,
miRNA
3. RNA Pol III- Produces tRNA, mRNA (5S),
SRPRNA

7. In Prokaryotes & Eukaryotes
Prokaryotes
 Occurs in cytoplasm
 Coupled transcription &
translation
 No definite phase of
occurrence
 A single RNAP synthesizes
mRNA, tRNA & rRNA
 No initiation factors
required
 Polycistronic
Eukaryotes
 Occurs in nucleus
 No coupling of
transcription & translation
 Occurs in the G1 & G2
phases
 RNAP I, II & III synthesize
rRNA, mRNA & tRNA
 TFIIA, TFIIB, TFIID, TFIIE,
TFIIF, TFIIH recognize TATA
box
 Monocistronic

8. Polycistronic & Monicistronic
Monocistronic
 An mRNA molecule is said to
be monocistronic if it contains
genetic information to
translate only a single protein.
 In the end only one
polypeptide chain coding for
one protein is obtained from
a single gene with an operator
& promoter region.
Polycistronic
 Polycistronic mRNA contains
information for several genes
which are translated into
several proteins.
 mRNA contains several ORFs
(open reading frames), each
of which is translated in
proteins. The coding is
grouped and all of the genes
are translated together with a
common promoter &
operator region (like in
operons)

9. Pre-Initiation
First step of transcription.
The Pre-Initiation Complex (PIC) includes
RNA Polymerase II and six transcription
factors- TFIIA, TFIIB, TFIID, TFIIE, TFIIF,
TFIIH
Other co-activators and chromatin
remodeling complexes also comprise of PIC

10. Pre-Initiation (contd.)
• Can be summarized in 3 steps:
1. TATA Binding Protein (TBP) is a subunit of TFIID
and binds to the promoter, creating a sharp bend.
2. TBP-TFIIA interact; TBP-TFIIB interact; TFIIB-TFIIF
interact & TFIIF recruits RNA Pol II; TFIIE
joins the group and recruits TFIIH
3. Subunits within TFIIH that
have ATPase and helicase activity create
negative superhelical tension in the DNA.

11. Initiation
1. Negative superhelical tension causes approximately
one turn of DNA to unwind and form the transcription
bubble. Promoter melting requires hydrolysis by ATP
and is mediated by TFIIH.
2. TFIIH pulls the double stranded DNA into the cleft of
RNA Polymerase and helps in transition from closed to
open state. The two strands get separated.

12. Initiation

13. Abortive Initiation
• Before entering elongation phase, The
polymerase may terminate prematurely.
• This produces a truncated polypeptide chain.
• Many cycles of abortive initiation may occur
before actually producing a growing polypeptide
chain.
• This helps in providing a scrunching kind of
motion.

14. Elongation
• The polypeptide chain is elongated with the help of
Elongation Factors.
• RNA Pol conveniently adds nucleotides to the 3’ end.
The template strand for this is known as the sense
strand and the other anti-sense strand.
• There are different classes of elongation factors.
Some factors can increase the overall rate of
transcribing, some can help the polymerase through
transient pausing sites, and some can assist the
polymerase to transcribe through chromatin

15. Transcription Fidelity
• RNA polymerases select correct nucleoside
triphosphate (NTP) substrate to prevent
transcription errors. Only the NTP which
correctly base pairs with the coding base in the
DNA is admitted to the active center.
• RNA polymerase performs two known
proofreading functions to detect and remove
misincorporated nucleotides: pyrophosphorylytic
editing and hydrolytic editing

16. Pausing and Backtracking
• RNA polymerase does not transcribe through a gene
at a constant pace. Rather it pauses periodically at
certain sequences, sometimes for long periods of
time before resuming transcription.
• Promoter-proximal pausing during early elongation
is a commonly used mechanism for regulating genes
poised to be expressed rapidly or in a coordinated
fashion. The blockage is released once the
polymerase receives an activation signal

17. Termination
• Two Types:
1. Factor Dependent
2. Factor Independent
• Factor dependent requires Termination Factors
along with RNA Pol I.
• Factor Independent termination can be done
by RNA Pol III. A stretch of Thymines along a
hair pin loop causes disintegration of
complexes.

18. Post Transcriptional Modifications:
1. RNA Splicing:
• Introns are removed
• Exons are joined
• Small nuclear riboproteins (snRNP) like
spliceosomes help catalyse the reaction.
• Self splicing introns also exist.
• Mainly found in eukaryotes

19. Post Transcriptional Modifications
2. 5’ end Capping
• A guanine nucleotide linked to the 5’ end
triphosphate
3. Polyadenylation
• Poly Adenine units added to 3’ end of the
Ribonucleotide chain.

20. Inhibition of Transcription
• Most antibiotics are transcription inhibitors and are
useful against bacterial and fungal pathogens.
• They inhibit action by binding to RNA Polymerases,
DNA helicases, DNA topoisomerases or by producing
free radicals affecting transcription
• For Eg, Rifampicin binds to the β Subunit of RNA
Polymerase

21. “I placed some of the DNA on the ends of my fingers and rubbed them
together. The stuff was sticky. It began to dissolve on my skin. 'It's
melting -- like cotton candy.'
'Sure. That's the sugar in the DNA,' Smith said.
'Would it taste sweet?'
'No. DNA is an acid, and it's got salts in it. Actually, I've never tasted it.'
Later, I got some dried calf DNA. I placed a bit of the fluff on my tongue. It
melted into a gluey ooze that stuck to the roof of my mouth in a blob. The
blob felt slippery on my tongue, and the taste of pure DNA appeared. It
had a soft taste, unsweet, rather bland, with a touch of acid and a hint of
salt. Perhaps like the earth's primordial sea. It faded away.
Page 67, in Richard Preston's biographical essay on Craig Venter, "The
Genome Warrior" (originally published in The New Yorker in 2000). ”
― Timothy Ferris,The Best American Science Writing 2001