All eukaryotes have at least three different RNA polymerase (Pol I, II,and III; and plants have a Pol IV & a Pol V). In addition, whereas bacteria require only one additional initiation factor (σ), several initiation factors are required for efficient and promoter-specific initiation in eukaryotes. These are called the general transcription factors (GTFs)

1. Dr. Pawan Kumar Kanaujia
Assistant Professor
Molecular Biology (Theory)
Eukaryotic Transcription

6. TRANSCRIPTION IN EUKARYOTES
Whereas bacteria have only one RNA polymerase, all eukaryotes have at
least three different ones (Pol I, II,and III; and plants have a Pol IV & a Pol V).
In addition, whereas bacteria require only one additional initiation factor (σ),
several initiation factors are required for efficient and promoter-specific
initiation in eukaryotes. These are called the general transcription factors (GTFs).
The Subunits of RNA Polymerases

7. RNA Polymerase II Core Promoters Are Made Up of Combinations
of Different Classes of Sequence Element
The eukaryotic core promoter refers to the minimal set of sequence
Elements required for accurate transcription initiation by the Pol II machinery.
A core promoter is typically ~40–60 nucleotides long, extending either
upstream or downstream from the transcription start site.
These are the TFIIB recognition element (BRE), the TATA element (or box),
the initiator (Inr), and the downstream promoter elements (known as DPE,
DCE, and MTE).
Typically, a promoter includes some subset of these elements.
Thus, for example, promoters typically have either a TATA element or a
DPE element, not both.
Often, a TATA-containing promoter also contains a DCE. The Inr is the
most common element, found in combination with both TATA and DPEs.

8. Beyond—and typically upstream of—the core promoter, there are other
sequence elements required for accurate and efficient transcription in vivo.
Together, these elements constitute the regulatory sequences and can be
grouped into various categories, reflecting their location, and the organism.
These elements include promoter proximal elements, upstream activator
sequences (UASs), enhancers, and a series of other elements called
silencers, boundary elements, and insulators.
 All of these DNA elements bind regulatory proteins
(activators and repressors)

9. Pol II core promoter
Fig 1: The figure shows the positions of various DNA elements relative to the
transcription start site (indicated by the arrow above the DNA). These
elements, described in the text, are as follows: (BRE) TFIIB recognition
element; (TATA) TATA box; (Inr) initiator element; (DPE) downstream
promoter element; and (DCE) downstream core element. Another element,
MTE (motif ten element), described in the text, is not shown in this figure
but is located just upstream of the DPE. Also shown are the consensus
sequences for each element and (above) the name of the general
transcription factor that recognizes each element.

10. RNA Polymerase II Forms a Preinitiation Complex with General
Transcription Factors at the Promoter
The general transcription factors collectively perform the functions as
Performed by σ in bacterial transcription. Thus, the general transcription
factors help polymerase bind to the promoter and melt the DNA
(comparable to the transition from the closed to open complex in the
bacterial case).
They also help polymerase escape from the promoter and embark
on the elongation phase. The complete set of general transcription factors
and polymerase, bound together at the promoter and poised (rediness for
action) for initiation, is called the preinitiation complex.
Pol II promoters contain a so-called TATA element (some -30 bp upstream
of the transcription start site), where preinitiation complex formation
begins.
The TATA element is recognized by the general transcription factor called
TFIID.
The component of TFIID that binds to the TATA DNA sequence is called
TBP (TATA-binding protein).

11. The other subunits in this complex are called TAFs (TBP-associated factors).
TAFs recognize other core promoter elements such as the Inr, DPE, and
DCE, although the strongest binding is between TBP and TATA.
Thus, TFIID is a critical factor in promoter recognition and preinitiation
complex establishment.
TBP–DNA complex provides a platform to recruit other general
transcription factors and polymerase itself to the promoter.
In vitro, these proteins assemble at the promoter in the following order
(Fig 1): TFIIA, TFIIB, TFIIF together with polymerase, and then TFIIE and
TFIIH.
Formation of the preinitiation complex containing these components is
followed by promoter melting.
In eukaryotes requires hydrolysis of ATP and is mediated by TFIIH.

12. Fig 2: The stepwise assembly of the Pol II
preinitiation complex is shown here and
described in detail in the previous or above
slide. Once assembled at the promoter, Pol
II leaves the preinitiation complex upon
addition of the nucleotide precursors
required for RNA synthesis and after
phosphorylation of serine resides within
the enzyme’s “tail”. The tail contains
multiple repeats of the heptapeptide
sequence: Tyr-Ser-Pro-Thr-Ser- Pro-Ser
Transcription initiation by RNA Pol II

13. Promoter Escape Requires Phosphorylation of the Polymerase “Tail”
During abortive initiation, the polymerase synthesizes a series of short
transcripts.
In eukaryotes, promoter escape involves two steps not seen in bacteria:
one is ATP hydrolysis (in addition to the earlier ATP hydrolysis needed for
DNA melting), and the other is phosphorylation of the polymerase.
The large subunit of Pol II has a carboxy-terminal domain (CTD), which is
referred to as the “tail” (see Fig 2). The CTD contains a series of repeats
of the heptapeptide sequence: Tyr-Ser-Pro-Thr-Ser-Pro-Ser.
There are 27 of these repeats in the yeast Pol II CTD,
32 in the worm, 45 in the fly Drosophila and 52 in humans.
Indeed, the number of repeats seems to correlate with the complexity
of the genome. Each repeat contains sites for phosphorylation by specific
kinases, including one that is a subunit of TFIIH.
When Pol II recruited to the promoter initially unphosphorylated tail,
but the species found in the elongation complex bears multiple
phosphoryl groups on its tail.
Phosphorylation state of the CTD of Pol II controls subsequent steps—
elongation and even processing of the RNA—as well.

14. The Other General Transcription Factors Also Have Specific Roles in
Initiation
Table: The General Transcription Factors of RNA Polymerase II

15. TAFs :- TBP is associated with about 10 TAFs. Two of the TAFs bind DNA
elements at the promoter, for example, the initiator element (Inr) and the
downstream promoter elements (Fig 1).
TFIIB:- This protein, a single polypeptide chain, enters the preinitiation
complex after TBP (Fig 2). The crystal structure of the ternary complex of
TFIIB–TBP–DNA shows specific TFIIB–TBP and TFIIB–DNA contacts
(Fig 4). These include base-specific interactions with the major groove
upstream (to the BRE) (Fig 1) and the minor groove downstream of
the TATA element.
TFIIF :- This two-subunit (in humans) factor associates with Pol II and
is recruited to the promoter together with that enzyme (and other factors).
Binding of Pol II–TFIIF stabilizes the DNA–TBP–TFIIB complex and is required
before TFIIE and TFIIH are recruited to the preinitiation complex (Fig2).
TFIIE and TFIIH:- TFIIE, which, like TFIIF, consists of two subunits, binds
next and has roles in the recruitment and regulation of TFIIH. TFIIH controls
the ATP-dependent transition of the preinitiation complex to the open
complex.

16. In Vivo, Transcription Initiation Requires Additional Proteins,
Including the Mediator Complex
One reason for these additional requirements is that the DNA template
in vivo is packaged into chromatin. This condition complicates binding to the
promoter of polymerase and its associated factors.
Transcriptional regulatory proteins called activators help recruit
polymerase to the promoter, stabilizing its binding there.
This recruitment is mediated through interactions between DNA-bound
activators, chromatin-modifying and -remodeling factors, and parts of the
transcription machinery.
Mediator is associated with the basic transcription machinery,
most likely touching the CTD “tail” of the large polymerase subunit
through one surface, while presenting other surfaces for interaction with
DNA-bound activators.

17. Assembly of the preinitiation complex in the presence of Mediator,
nucleosome modifiers and remodelers, and transcriptional activators
Fig 5: Tanscriptional activators bound to sites near the gene recruit
nucleosome-modifying and –remodeling complexes and the Mediator
complex, which together help form the preinitiation complex.

18. A New Set of Factors Stimulates Pol II Elongation and RNA Proofreading
Once polymerase has escaped the promoter and initiated transcription,
it shifts into the elongation phase, as we have discussed. This transition
involves the Pol II enzyme shedding most of its initiation factors—for
example, the general transcription factors and Mediator. In their place,
another set of factors is recruited. Some of these (such as TFIIS and SPT5)
are elongation factors (i.e., factors that stimulate elongation).
In this case, however, the factors favor the phosphorylated form of
the CTD. Thus, phosphorylation of the CTD leads to an exchange of
initiation factors for those factors required for elongation and
RNA processing.
Together, these features allow the tail to bind several components of
the elongation and processing machinery and deliver them to the
emerging RNA.
Various proteins are thought to stimulate elongation by Pol II. One of
these, the kinase P-TEFb, is recruited to polymerase by transcriptional
activators.

19. Once bound to Pol II, this protein phosphorylates the serine residue
at position 2 of the CTD repeats.
That phosphorylation event correlates with elongation (Fig 6)
In addition, P-TEFb phosphorylates and thereby activates another
protein, called SPT5, itself an elongation factor. Finally, TAT-SF1, yet
another elongation factor, is recruited by P-TEFb.

20. Fig 6:-RNA processing enzymes are recruited by the CTD tail of polymerase

21. Fig 6: (a) Various factors involved in RNA processing recruited by the CTD
tail of polymerase. Different factors are recruited depending on the
phosphorylation state of the tail. Those factors are then transferred to the
RNA as they are needed (see next section in text).
(b) A schematic of the tail, with the sequence of one copy of the
heptapeptide repeat shown in the top line. The positions of serine residues
that get phosphorylated are indicated in lines 2 and 3. Phosphorylation of
serine at position 5 is seen upon promoter escape and is associated with
recruitment of capping factors, where as phosphorylation of serine at
position 2 is seen during elongation and is associated with recruitment
of splicing factors. Recruitment of factors involved in elongation of
transcription and in RNA processing overlaps. Thus, elongation factor
hSPT5 is recruited to the tail phosphorylated on Ser-5.

22. Elongating RNA Polymerase Must Deal with Histones in Its Path
As with initiation of transcription, elongation also takes place in the
presence of histones, because the DNA template is incorporated into
nucleosomes.
Factor called FACT (facilitates chromatin transcription) was identified in
human makes transcription on chromatin templates much more efficient.
FACT is a heterodimer of two well conserved proteins, Spt16 and SSRP1.
Nucleosomes are octomers, made up of H2A, H2B, H3,and H4 histone
subunits and DNA.
These histones are arranged in two modules: the H2A.H2B dimers and
the H3.H4 tetramer.
Ahead of a transcribing RNA polymerase, FACT removes one H2A.H2B
dimer. This allows polymerase to pass that nucleosome .
FACT also has histone chaperone activity, which allows it to restore
the H2A.H2B dimer to the histone hexamer immediately behind the
Processing polymerase.
In this way, FACT allows polymerase to elongate and at the same time
maintains the integrity of the chromatin through which the enzyme is
transcribing.

23. A model for FACT-aided elongation through nucleosomes
Fig 8:- Step 1- FACT, shown as the heterodimer of Spt16 and SSRP1, is able to
dismantle nucleosomes ahead of the transcribing RNA polymerase
and reassemble them behind.
Step 2- Specifically, it removes the H2A.H2B dimer. SPT6 binds histone H3
and is believed to aid in nucleosome reassembly.

24. Elongating Polymerase Is Associated with a New Set of Protein
Factors Required for Various Types of RNA Processing
These processing events include capping of the 5’ end of the RNA, splicing,
and polyadenylation of the 3’ end of the RNA. The most complicated of
these is splicing—the process whereby non-coding introns are removed
from RNA to generate the mature mRNA.
Here we consider the other two processes—capping and
polyadenylating the transcript.
In one case, for example, an elongation factor mentioned above (SPT5)
also helps to recruit the 5’-capping enzyme to the CTD tail of polymerase
(phosphorylated at serine position 5) (Fig 6b).The hSPT5 stimulates the
5’-capping enzyme activity.
In another case, elongation factor TAT-SF1 recruits components of the
splicing machinery to polymerase with a Ser-2 phosphorylated tail (Fig 6b).
Thus, elongation, termination of transcription, and RNA processing are
interconnected, presumably to ensure their proper coordination.

25. The 5’ cap is created in three enzymatic steps, as detailed in Fig 9
and described in detail in the legend.
In the first step, a phosphate group is removed from the 5’ end of the
transcript.
Then, in the second step, the GMP moiety is added.
In the final step, that nucleotide is modified by the addition of a methyl
group. The RNA is capped as soon as it emerges from the RNA-exit channel
of polymerase.
This happens when the transcription cycle has progressed only as far as
the transition from the initiation to elongation phases.
After capping, dephosphorylation of Ser-5 within the tail repeats
may be responsible for dissociation of the capping machinery, and further
phosphorylation (this time of Ser-2 within the tail repeats) causes
recruitment of the machinery needed for RNA splicing (Fig 6b)

26. Fig 9:- In the first step, the ϒ-phosphate at the
5’ end of the RNA is removed by an enzyme
called RNA triphosphatase (the initiating
nucleotide of a transcript initially retains
its α-, β-, and ϒ phosphates).
In the next step, the enzyme
guanylyltransferase adds a GMP moiety to
the resulting terminal β-phosphatase.
This is a two-step process: first,
an enzyme–GMP complex is generated
from GTP with release of the β- and
ϒ -phosphates of that GTP, and then the
GMP from the enzyme is transferred
to the β-phosphate of the 5’ end of the RNA.
Once this linkage is made, the newly added
guanine and the purine at the original 5’
end of the mRNA are further modified by
the addition of methyl groups by
methyltransferase. The resulting 5’ cap
structure subsequently recruits the ribosome
to the mRNA for translation to begin.
The structure and formation
of the 5’RNA cap

27. The final RNA processing event, polyadenylation of the 3’ end of the
mRNA, is intimately linked with the termination of transcription (Fig 10).
Just as with capping and splicing, the polymerase CTD tail is involved
in recruiting some of the enzymes necessary for polyadenylation (Fig 6).
Once polymerase has reached the end of a gene, it encounters specific
sequences that, after being transcribed into RNA, trigger the transfer
of the polyadenylation enzymes to that RNA, leading to four events:
Cleavage of the message;
 addition of many adenine residues to its 3’ end;
Degradation of the RNA remaining associated with RNA pol
by a 5’-to-3’ ribonuclease; and,
subsequently, termination of transcription.
This series of events unfolds as follows.

28. Two protein complexes are carried by the CTD of polymerase as it
approaches the end of the gene: CPSF (cleavage and polyadenylation
specificity factor)and CSTF (cleavage stimulation factor).
The sequences that, once transcribed into RNA, trigger transfer of
these factors to the RNA are called poly-A signals, and their operation
is shown in Fig 10. Once CPSF and CSTF are bound to the RNA, other
proteins are recruited as well, leading initially to RNA cleavage and
then polyadenylation.
Polyadenylation is mediated by an enzyme called poly-A polymerase,
which adds approximately 200 adenines to the RNA’s 3’ end produced by
the cleavage.
This enzyme uses ATP as a precursor and adds the nucleotides
using the same chemistry as RNA polymerase. But it does so without
a template. Thus, the long tail of As is found in the RNA but not the
DNA.
The mature mRNA is then transported from the nucleus.

29. Polyadenylation
and termination
Fig 10:-The various steps in this
Process are described in the above text.

30. Transcription Termination Is Linked to RNA Destruction
by a Highly Processive RNase
Polyadenylation is linked to termination, although exactly how is still not
quite clear. Recently, however, an enzyme that degrades the second RNA
as it emerges from the polymerase has been identified, and this enzyme
may itself trigger termination. This is called the torpedo model of
termination (Fig 11a).
The free end of the second RNA is uncapped and thus can be
distinguished from genuine transcripts. This new RNA is recognized by an
RNase called, in yeast, Rat1 (in humans, Xrn2) that is loaded onto the end
of the RNA by another protein (Rtt103) that binds the CTD of RNA pol.
The Rat1 enzyme is very processive and quickly degrades the RNA in a
5’-to-3’ direction, until it catches up to the still-transcribing polymerase
from which the RNA is being spewed.
Although the torpedo model for termination is now the favored one,
there is an alternative called the allosteric model (Fig 11b).
According to this model, termination depends on a conformational
change in the elongating polymerase that reduces the processivity of the
enzyme leading to spontaneous termination soon afterward.

31. Fig 11:- Models of termination: torpedo and allosteric

32. Fig 11:- there are two proposed models for how transcription by
eukaryotic RNA Pol II terminates after transcribing a gene. In the figure,
the poly-A site is marked by the light green stretch in the DNA and is
located just downstream from the gene. It is also light green in the
transcript. (The dotted green line) Degraded transcript.
(a) In the torpedo model, RNA transcribed downstream from the
poly-A site is attacked by the 5’-to-3’ RNase (the torpedo), which is
loaded onto this transcript from polymerase itself. When this exonuclease
catches up with polymerase, it triggers dissociation from the DNA template
and termination of transcription.
(b) In the allosteric model, the polymerase is highly processive within the
gene, and then, once the poly-A signal is passed, becomes less processive.
This alteration could be due to a modification or a conformational change.
Even in the allosteric model, the second RNA would be degraded by the
RNase, but that would not be the cause of termination. In this case, RNA
degradation is not shown in the figure to emphasize the different
mechanisms of termination in these two models.

33. Thank you