Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of transportable complementary RNA replica.

1. Eukaryotic Transcription
Prasanna R Kovath
Assistant Professor
Department of Biotechnology

2. • In bacteria, transcription takes place on a DNA template, whereas in
eukaryotes, transcription takes place on a chromatin template.
• A second major difference is that the bacterial RNA polymerase, with
its sigma factor subunit, can read the DNA sequence to find and bind
to its promoter.
• A eukaryotic RNA polymerase cannot read the DNA.
• Initiation at eukaryotic promoters therefore involves a large number of
factors that must prebind to a variety of cis-acting elements and other
factors already bound to the DNA before the RNA polymerase can
bind. These factors are called basal transcription factors.

3. • The RNA polymerase then binds to this basal transcriptionfactor/DNA
complex.
• This binding region is defined as the core promoter, the region
containing all the binding sites necessary for RNA polymerase to
bind and function.
• RNA polymerase itself binds around the start point of transcription,
but does not directly contact the extended upstream region of the
promoter.

4. • Bacteria have a single RNA polymerase that transcribes all three major
classes of genes, transcription in eukaryotic cells is divided into three
classes. Each class is transcribed by a different RNA polymerase:
• RNA polymerase I transcribes 18S/28S rRNA.
• RNA polymerase II transcribes mRNA and a few small RNAs.
• RNA polymerase III transcribes tRNA, 5S ribosomal RNA, and some
other small RNAs.

5. • Basal transcription factors are needed for initiation, but most are not
required subsequently. For the three eukaryotic RNA polymerases, the
transcription factors, rather than the RNA polymerases themselves, are
responsible for recognizing the promoter DNA sequence.
• The basal factors together with RNA polymerase constitute the basal
transcription apparatus.
• The basal factors join with RNA polymerase II to form a complex
surrounding the start point, and they determine the site of
initiation.

6. • The promoters for RNA polymerases I and II are (mostly) upstream of
the start point, but a large number of promoters for RNA polymerase
III lie downstream (within the transcription unit) of the start point.
• Upstream : (5' to) is in the direction from which the polymerase (or
ribosome) has come.
• Downstream : (or 3' to) is in the direction of transcription or
translation
• Each promoter contains characteristic sets of short conserved
sequences that are recognized by the appropriate class of basal
transcription factors.
• RNA polymerases I and III each recognize a relatively restricted set of
promoters, and thus rely upon a small number of accessory factors.

7. • All RNA polymerase II promoters have sequence elements close to the
start point that are bound by the basal apparatus and the polymerase to
establish the site of initiation.
• Other sequences farther upstream or downstream, called enhancer
sequences, determine whether the promoter is expressed, and if
expressed, whether this occurs in all cell types or is cell type specific.
• An enhancer is another type of site involved in transcription and is
identified by sequences that stimulate initiation, but that are located a
variable distance from the core promoter

9. • A regulatory site that binds more negative regulators than positive
regulators to control transcription is called a silencer.
• Promoters that are constitutively expressed and needed in all cells
(their genes are sometimes called housekeeping genes)
• Enhancers do not need to be near the promoter. They can be upstream,
inside a gene, or beyond the end of a gene, and their orientation
relative to the gene does not matter.
• Proteins bound at enhancer elements interact with proteins bound at
promoter elements via DNA looping, very often through intermediates
called coactivators.

10. • A transcription factor (TF) (or sequence-specific DNA-binding
factor) is a protein that controls the rate
of transcription of genetic information from DNA to messenger RNA,
by binding to a specific DNA sequence.
• The function of TFs is to regulate—turn on and off—genes in order to
make sure that they are expressed in the right cell at the right time and
in the right amount throughout the life of the cell and the organism.
• TFs work alone or with other proteins in a complex, by promoting (as
an activator), or blocking (as a repressor) the recruitment of RNA
polymerase (the enzyme that performs the transcription of genetic
information from DNA to RNA) to specific genes.
• A defining feature of TFs is that they contain at least one DNA-
binding domain (DBD), which attaches to a specific sequence of DNA
adjacent to the genes that they regulate.
• TFs are grouped into classes based on their DBDs

11. • Two general types of transcription factors have been defined. General
transcription factors (GTFs) are involved in transcription from all
polymerase II promoters and therefore constitute part of the basic
transcription machinery.
• Many of these GTFs do not actually bind DNA, but rather are part of
the large transcription preinitiation complex that interacts with RNA
polymerase directly. The most common GTFs
are TFIIA, TFIIB, TFIID , TFIIE, TFIIF, and TFIIH.
• The preinitiation complex (PIC) binds to promoter regions of DNA
upstream to the gene that they regulate.
• Additional transcription factors bind to DNA sequences that control
the expression of individual genes and are thus responsible for
regulating gene expression.

12. • Five general transcription factors are required for initiation of
transcription by RNA polymerase II in reconstituted in vitro systems .
• The promoters of many genes transcribed by polymerase II contain a
sequence similar to TATAA 25 to 30 nucleotides upstream of the
transcription start site.
• This sequence (called the TATA box) resembles the -10 sequence
element of bacterial promoters, and the results of introducing
mutations into TATAA sequences have demonstrated their role in the
initiation of transcription.
Organization of a eukaryotic protein-coding gene region

13. • The first step in formation of a transcription complex is the binding of a
general transcription factor called TFIID to the TATA
box (TF indicates transcription factor; II indicates polymerase II).
• TFIID is itself composed of multiple subunits, including the TATA-
binding protein (TBP), which binds specifically to the TATAA
consensus sequence, and 10-12 other polypeptides, called TBP-
associated factors (TAFs).
• TBP then binds a second general transcription factor (TFIIB) forming a
TBP-TFIIB complex at the promoter
• TFIIB in turn serves as a bridge to RNA polymerase, which binds to the
TBP-TFIIB complex in association with a third factor, TFIIF.
• Following recruitment of RNA polymerase II to the promoter, the
binding of two additional factors (TFIIE and TFIIH) is required for
initiation of transcription.

14. Formation of a polymerase II transcription complex
Many polymerase II promoters have a TATA box (consensus sequence
TATAA) 25 to 30 nucleotides upstream of the transcription start site. This
sequence is recognized by transcription factor TFIID, which consists of
the TATA-binding protein (TBP) and TBP-associated factors (TAFs). TFIIB(B)
then binds to TBP, followed by binding of the polymerase in association
with TFIIF(F). Finally, TFIIE(E) and TFIIH(H) associate with the complex.
Transcription initiation by RNA
polymerase II in a eukaryotic
cell

15. • In addition to a TATA box, the promoters of many genes transcribed
by RNA polymerase II contain a second important sequence element
(an initiator, or Inr, sequence) that spans the transcription start site.
• Moreover, some RNA polymerase II promoters contain only an Inr
element, with no TATA box.
• Initiation at these promoters still requires TFIID (and TBP), even
though TBP obviously does not recognize these promoters by binding
directly to the TATA sequence.
• Instead, other subunits of TFIID (TAFs) appear to bind to the Inr
sequences. This binding recruits TBP to the promoter, and TFIIB,
polymerase II, and additional transcription factors then assemble as
already described.
• TBP thus plays a central role in initiating polymerase II transcription,
even on promoters that lack a TATA box.

16. Eukaryotic Transcription Initiation: A generalized promoter of a gene transcribed
by RNA polymerase II is shown. Transcription factors recognize the promoter,
RNA polymerase II then binds and forms the transcription initiation complex.

17. • First, two subunits of TFIIH are helicases, which may unwind DNA around the
initiation site.
• Another subunit of TFIIH is a protein kinase that phosphorylates repeated sequences
present in the C-terminal domain of the largest subunit of RNA polymerase II.
• a protein tail of RNA polymerase II subunit called carboxyl tail domain (CTD) CTD
is located near the region of the new RNA synthesized from the polymerase.
• After the phosphorylation of CTD with one of the general transcription factors,
the start phase ends up and the elongation phase begins.
• Phosphorylation of these sequences is thought to release the polymerase from its
association with the initiation complex, allowing it to proceed along the template as
it elongates the growing RNA chain.
• The CTD contains a series of repeats of heptapeptide sequence: Tyr-Ser-Pro-Thr-
Ser-Pro-Ser.
• Phosphorylation of the CTD tail is needed to release RNA polymerase II from the
promoter and transcription factors so that it can make the transition to the elongating
form.

18. RNA polymerase motions during promoter melting
On a linear template, ATP
hydrolysis,TF11E, and the
helicase activity of TF11H
(provided by the XPB and XPD
subunits) are required for
polymerase movement. This
requirement is bypassed with a
supercoiled template. This
suggests that TF11E and TF11H
are required to melt DNA to
allow polymerase movement to
begin. The helicase activity of
the XPB subunit ofTF11H is
responsible for the
actual melting of DNA.

19. PROMOTER MELTING
• The denaturation or separation of the two strands of DNA of the
promoter region on binding a 3′→5′ DNA helicase subunit of
transcription factor TFIIH.ATPase activity (ATP hydrolysis).
• Abortive initiation, also known as abortive transcription,
• Is an early process of genetic transcription in which RNA polymerase
binds to a DNA promoter and enters into cycles of synthesis of short
mRNA transcripts which are released before the transcription complex
leaves the promoter.
• Abortive initiation refers to the repetitive synthesis and release of
short nascent RNAs by RNA polymerase.

20. ELONGATION
• RNA polymerase II now starts moving along the DNA template,
synthesizing RNA, that is, the process enters the elongation phase.
• RNA synthesis occurs in the 5’ → 3’ direction with the RNA polymerase
catalyzing a nucleophilic attack by the 3-OH of the growing RNA chain
on the alpha-phosphorus atom on an incoming ribonucleoside 5-
triphosphate.
• The RNA molecule made from a protein-coding gene by RNA polymerase
II is called a primary transcript.
• For RNA synthesis to occur, the transcription machinery needs to move
histones out of the way every time it encounters a nucleosome.
• This is accomplished by a special protein complex called FACT, which
stands for “facilitates chromatin transcription.” This complex pulls
histones away from the DNA template as the polymerase moves along it.
• Once the pre-mRNA is synthesized, the FACT complex replaces the
histones to recreate the nucleosomes.

21. ELONGATION FACTORS
• Among the proteins recruited to polymerase are elongation factors,
thus called because they stimulate transcription elongation.
• 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.
• TFIIS & hSPT5, known as elongation factors. This factors stimulate
elongation and also required for RNA processing.
• These factors also favor the phosphorylated form of CTD.
• The phosphorylation of CTD leads to an exchange of initiation factors
with elongation factors.

22. • Various proteins are thought to stimulate elongation by Pol II.
• The protein P-TEFb stimulates elongation in 3 separate steps.
• This protein bound to Pol II and phosphorylates the serine residue at
position 2 of the CTD repeats .
• This P-TEFb also activates another protein, called hSPT5 which is an
elongation factor.
• At last, this P-TEFb activates one another elongation factor called
TAT-SF1.
• The elongation factors can exert their effects through a number of
distinct mechanisms, which include controlling the rate of elongation
or the processivity of Pol II, facilitating transcription through
nucleosomes, or by reactivating Pol II that has become arrested.

23. • RNA Polymerases unwind the double stranded DNA ahead of them and
allow the unwound DNA behind them to rewind. As a result, RNA strand
synthesis occurs in a transcription bubble of about 25 unwound DNA
basebairs. Only about 8 nucleotides of newly-synthesized RNA remain
basepaired to the template DNA. The rest of the RNA molecules falls off
the template to allow the DNA behind it to rewind.
• RNA Polymerases use the DNA strand below them as a template to direct
which nucleotide to add to the 3′ end of the growing RNA strand at each
point in the sequence. The RNA Polymerase travels along the template
DNA one nucleotide at at time. Whichever RNA nucleotide is capable of
basepairing to the template nucleotide below the RNA Polymerase is the
next nucleotide to be added. Once the addition of a new nucleotide to the 3′
end of the growing strand has been catalyzed, the RNA Polymerase moves
to the next DNA nucleotide on the template below it. This process
continues until transcription termination occurs.

24. Transcription Elongation Produces Superhelical Tension in
DNA
• Once it has initiated transcription, RNA polymerase does not proceed
smoothly along a DNA molecule; rather it moves jerkily, pausing at some
sequences and rapidly transcribing through others.
• Elongation factors typically associate with RNA polymerase shortly after
initiation has occurred and help polymerases to move through the wide
variety of different DNA sequences that are found in genes.
• Eukaryotic RNA polymerases must also contend with chromatin structure
as they move along a DNA template.
• chromatin remodeling complexes may move with the polymerase In
addition, some elongation factors associated with eukaryotic RNA
polymerase facilitate transcription through nucleosomes without requiring
additional energy. It is not yet understood how this is accomplished, but
these proteins may help to dislodge parts of the nucleosome core as the
polymerase transcribes the DNA of a nucleosome.

25. • There is yet another barrier to elongating polymerases, DNA
supercoiling
• DNA supercoiling represents a conformation that DNA will adopt in
response to superhelical tension; conversely, creating various loops or
coils in the helix can create such tension.
• Superhelical tension is also created as RNA polymerase moves along a
stretch of DNA that is anchored at its ends.
• As long as the polymerase is not free to rotate rapidly a moving
polymerase generates positive superhelical tension in the DNA in front
of it and negative helical tension behind it.

26. • For eukaryotes, this situation is thought to provide a bonus: the
positive superhelical tension ahead of the polymerase makes the DNA
helix more difficult to open, but this tension should facilitate the
unwrapping of DNA in nucleosomes, as the release of DNA from
the histone core helps to relax positive superhelical tension.
• In eukaryotes, DNA topoisomerase enzymes rapidly remove this
superhelical tension.
• In bacteria, a specialized topoisomerase called DNA gyrase uses the
energy of ATP hydrolysis to pump supercoils continuously into the
DNA, thereby maintaining the DNA under constant tension. These
are negative supercoils, These negative supercoils are removed from
bacterial DNA whenever a region of helix opens, reducing the
superhelical tension.
• Thus helps the initiation of transcription by bacterial RNA polymerase,
that require helix opening

27. (A) For a DNA molecule with one free end (or a nick
in one strand that serves as a swivel), the DNA double
helix rotates by one turn for every 10 nucleotide pairs
opened.
(B) If rotation is prevented, superhelical tension is
introduced into the DNA by helix opening. One
way of accommodating this tension would be to
increase the helical twist from 10 to 11 nucleotide
pairs per turn in the double helix that remains in this
example; the DNA helix, however, resists such a
deformation in a springlike fashion, preferring to
relieve the superhelical tension by bending into
supercoiled loops. As a result, one DNA supercoil
forms in the DNA double helix for every 10
nucleotide pairs opened. The supercoil formed in this
case is a positive supercoil

28. • (C) Supercoiling of DNA is induced by a protein tracking through the
DNA double helix.
• The two ends of the DNA shown here are unable to rotate freely
relative to each other, and the protein molecule is assumed also to be
prevented from rotating freely as it moves.
• Under these conditions, the movement of the protein causes an excess
of helical turns to accumulate in the DNA helix ahead of the protein
and a deficit of helical turns to arise in the DNA behind the protein,

30. TERMINATION
• The termination of transcription is different for the different
polymerases. Unlike in prokaryotes, elongation by RNA polymerase II
in eukaryotes takes place 1,000–2,000 nucleotides beyond the end of
the gene being transcribed.
• The ribosomal rRNA genes transcribed by RNA Polymerase I contain
a specific sequence of basepairs (11 bp long in humans; 18 bp in mice)
that is recognized by a termination protein called TTF-1 (Transcription
Termination Factor for RNA Polymerase I.)
• This protein binds the DNA at its recognition sequence and blocks
further transcription, causing the RNA Polymerase I to disengage from
the template DNA strand and to release its newly-synthesized RNA.

31. • The protein-encoding, structural RNA, and regulatory RNA genes
transcribed by RNA Polymerse II lack any specific signals or sequences
that direct RNA Polymerase II to terminate at specific locations.
• RNA Polymerase II can continue to transcribe RNA anywhere from a few
bp to thousands of bp past the actual end of the gene. However, the
transcript is cleaved at an internal site before RNA Polymerase II finishes
transcribing.
• This releases the upstream portion of the transcript, which will serve as the
initial RNA prior to further processing (the pre-mRNA in the case of
protein-encoding genes.) This cleavage site is considered the “end” of the
gene.

32. • The remainder of the transcript is digested by a 5′-exonuclease (called
Xrn2 in humans) while it is still being transcribed by the RNA
Polymerase II.
• When the 5′-exonulease “catches up” to RNA Polymerase II by
digesting away all the overhanging RNA, it helps disengage the
polymerase from its DNA template strand, finally terminating that
round of transcription.
• The tRNA, 5S rRNA, and structural RNAs genes transcribed by RNA
Polymerase III have a not-entirely-understood termination signal.
• The RNAs transcribed by RNA Polymerase III have a short stretch of
four to seven U’s at their 3′ end. This somehow triggers RNA
Polymerase III to both release the nascent RNA and disengage from
the template DNA strand.

33. • Unlike in prokaryotes, elongation by RNA polymerase II in eukaryotes
takes place 1,000–2,000 nucleotides beyond the end of the gene being
transcribed.
• This pre-mRNA tail is removed during mRNA processing. RNA
polymerases I and III require termination signals. Genes transcribed by
RNA polymerase I contain a specific 18-nucleotide sequence that is
recognized by a termination protein.
• The process of termination in RNA polymerase III involves an mRNA
hairpin that causes the mRNA to be released.

35. RNA PROCESSING
• These RNA processing steps are tightly coupled to transcription
elongation by an ingenious mechanism.
• As discussed previously, a key step of the transition of RNA
polymerase II to the elongation mode of RNA synthesis is an
extensive phosphorylation of the RNA polymerase II tail, called the
CTD.
• This C-terminal domain of the largest subunit consists of a long
tandem array of a repeated seven-amino-acid sequence, containing two
serines per repeat that can be phosphorylated.
• This phosphorylation step not only dissociates the RNA polymerase II
from other proteins present at the start point of transcription, it also
allows a new set of proteins to associate with the RNA polymerase tail
that function in transcription elongation and pre-mRNA processing.

36. • As discussed next, some of these processing proteins seem to “hop”
from the polymerase tail onto the nascent RNA molecule to begin
processing it as it emerges from the RNA polymerase.
• Thus, RNA polymerase II in its elongation mode
can be viewed as an RNA factory that both:
• transcribes DNA into RNA
• processes the RNA it produces.