This presentation is about the transcription machinery that is required for the transcription in eukaryotes. The comparison between the transcription factors involved in prokaryotes and eukaryotes. The initiation of transcription and how it helps in producing a mRNA.

2. What is transcription??
• It is the synthesis of an mRNA molecule from a DNA template.
• All the cellular RNAs are synthesized from the DNA templates through the
process of transcription.
• DNA regions that can be transcribed into RNA are called structural genes.
• The template strand on which the RNA is synthesized is called as antisense
strand or nonsense strand.

3. Fig: Central dogma of life wherein DNA is converted into mRNA
and mRNA into proteins via transcription and translation.

4. • The coding strand is called as sense strand.
• The enzyme that catalyzes this RNA synthesis is RNA
polymerase and doesn’t needs a primer unlike replication.

7. TRANSCRIPTION IN EUKARYOTES
• Transcription in eukaryotes is undertaken by different RNA
polymerases.
• Eukaryotes have 3 polymerases: Pol I, II and III.
• Several initiation factors are required for efficient & promoter-
specific initiation in eukaryotes, and are called as general
transcription factors [GTFs].
• In vitro, the GTFs is required, together with Pol I, to initiate
transcription on a DNA template.

8. TRANSCRIPTION IN EUKARYOTES
• Sometimes the GTF’s are not sufficient to promote significant
expression. Rather, the additional factors are required such as
mediator complex, DNA binding, regulatory proteins and
chromatin modifying enzymes.
• In vitro, the GTFs is required, together with Pol II, to initiate
transcription on a DNA template.
• Sometimes the GTF’s are not sufficient to promote significant
expression. Rather, the additional factors are required such as
mediator complex, DNA binding, regulatory proteins and
chromatin modifying enzymes.

9. • Transcription in eukaryotic cells can be divided into three
classes. Each class is transcribed by a different RNA
polymerase:
• RNA polymerase I transcribes rRNA.
• RNA polymerase II transcribes mRNA.
• RNA polymerase III transcribes tRNA and other small RNA’s.
• In eukaryotic cells, for all the RNA polymerases , the factors
create a structure at the promoter to provide the target that is
recognized by the enzyme.
• E.g. RNA polymerase II the factors form a sizeable group
collectively called as basal factors.

10. What are transcription factors??
 Any protein that is needed for the initiation of transcription ,
but which is not itself part of RNA polymerase, is defined as a
transcription factor.
 They act by recognizing cis-acting sites on DNA.
 Binding of the transcription factors is not enough for the
transcription. It may recognize another factor, or RNA
polymerase, or it might get incorporated into an initiation
complex only in the presence of several other proteins.
 A protein is needed for the transcription to occur at a specific
promoter or set of promoters.

11. Transcription factors are needed for initiation.
What are basal factors??
• These are the factors that join with RNA polymerase II and form a complex
surrounding at the start point, and they determine the site of initiation.
Basal transcription apparatus:
 The basal factors and the RNA polymerase II together form the basal
transcription apparatus.
 The RNA polymerase II promoters have sequence elements close to the start
point that are bound by the basal apparatus and that establish the site of
initiation. RNA polymerase II and the general transcription factors are
needed to transcribe any promoter.
 The subunits of RNA polymerase II and the general transcription factors are
conserved among eukaryotes.

12. What is a promoter??
• The promoter is defined as the shortest region that
contains all the binding sites, that is those sites that can
support transcription at the normal efficiency and with the
proper control.
• For a eukaryotic mRNA it is basically the site at which the
transcription factors bind.
• The promoter contains several short <10bp sequence elements
that bind the transcription factors, dispersed over >200 bp.
• The promoters for RNA polymerase II are located upstream of
the start point and these help in initiating the transcription.

13. Fig: Organization of a gene in eukaryotic organism’s

14. Enhancers
• These are the type of sites that are involved in initiation. It can
be defined as the sequences that stimulate initiation but are
located at a considerable distance from the start point.
• The components of an enhancer are similar to those of the
promoter.
• They contain several closely arranged sequence elements that
bind transcription factors.
• The enhancer need not to be at the start point.
• The proteins that are bound to the enhancer also interact with
the proteins bound at promoter elements.

15. STEPS OF TRANSCRIPTION
• Transcription by RNA polymerase proceeds through a series of
well- defined steps which are grouped into 3 phases:
 Initiation
 Elongation and
 Termination.

16. RNA POLYMERASE II
• It is located in the nucleosome.
• It represents most of the remaining cellular activity and is
responsible for synthesizing heterogeneous nuclear RNA
(hnRNA), the precursor for mRNA.
• These are large proteins having molecular weight of > 500kD.
• RNA polymerase II requires transcription factors called
TFII[X] to initiate the transcription.
• The TATA box is a common component of RNA polymerase II
promoters and consists of A-T rich octamer located -25 bp
upstream of the start point.
• The core promoter for RNA polymerase II includes the InR
and either a TATA box or a DPE.

17. Fig: Steven Hahn 2004, Structure and mechanism of the RNA polymerase II
transcription machinery, Nature Structural &Molecular Biology 2004
DOI:10.1038/nsmb763

18. Core promoter:
• It refers to the minimal set of sequence elements required for accurate
transcription initiation by Pol II.
• Functions at low efficiency.
• A core promoter is about 40 nucleotides long, extending either upstream or
downstream of the transcription start site.
• There are 4 elements found in Pol II core promoter these are the :
1. TFIIB recognition element [BRE]
2. The TATA element
3. The initiator [Inr] and
4. The downstream promoter elements [DPE]
• Promoter includes only 2 or 3 of these 4 elements.
• A core promoter is present either with a TATA BOX and an Inr or with a Inr
and a DPE.

19. • Initiator- at the start point there is no extensive homology of
sequence, but there is a tendency for the first base of Mrna to
be A, flanked on either side by pyrimidines.
• This region is called as the initiator (Inr). This initiator can be
described in the form of Py2CAPy5. the initiator is between
positions -3 and +5.
• the TATA box and the TATA less box.
TATA BOX TATA LESS BOX
 These sequences are located
~25 bp upstream of the start point.
The core sequence is TATAA
which is followed by 3 more A-T bp.
 The promoters that do not contain a
TATA element are called as TATA-LESS
promoters.
If there is no TATA sequence then the
sequence called as the DPE ( downstream
promoter element is usually present.
Located at + 28 to +32.

20.  Core promoter with a tata box at -25 and at +1 there is
transcriptional start site.
 Coding strand sequence upto 100bp.
 Upstream from the tata box mny regulatory elements- Cis
acting elements.
 DNA sequences- that exert their effect only over a particular
gene. Eg- tata box, enhancers and silencers.
 Trans acting factors- that bind to cis-acting elements.

21. Proteins needed for transcription in eukaryotes
• RNA polymerase II: the enzyme that catalyzes the linkage of
ribonucleotides in the 5’ to 3’ direction, using DNA as a
template. Eukaryotic RNA polymerase II proteins are usually
composed of 12 subunits. The two largest subunits are
structurally similar to β and β’ subunits found in E.coli RNA
polymerase.

22. GENERAL TRANSCRIPTION FACTORS
IN EUKARYOTES

23. TFIID:
Composed of TATA-BINDING PROTEIN (TBP) and other
TBP-associated factors (TAFs). Recognizes the TATA box of
eukaryotic protein- encoding gene promoters.
TFIIB:
Binds to TFIID and then enables RNA polymerase II to bind
to the core promoter. Also promotes TFIIF binding. It is a
monomeric protein, slightly smaller than TBP.
TFIIF:
TFIIF A tetrameric complex with RNA polymerase II and
plays a role in its ability to bind to TFIIB and the core
promoter. Also plays a role in the ability of TFIIE and TFIIH to
bind to RNA polymerase II.
TFIIE:
TFIIE is a tetrameric complex that binds to the DNA and
creates a dock site for TFIIH.
TFIIH:
It is a multimeric factor containing 9 subunits. The binding
of TFIIH completes the assembly of the transcription pre-
initiation complex in vitro. It has multiple roles.

24. • TFIIH: certain subunits of the TFIIH act as helicases, break
the hydrogen bonds form the transcription bubble and promote
the formation of the open complex in this the DNA is melted
and the template site is bound at the polymerase active site .
Other subunits phosphorylate the carboxyl terminal domain of
RNA polymerase II, which releases its interaction with TFIIB,
TFIIE and TFIIH thereby allowing RNA polymerase II to
proceed to the elongation phase.

25. Fig: The pathway of transcription initiation and reinitiation for
RNA Pol II

26. PRE INITIATION COMPLEX FORMATION
• The GTFs help polymerase bind to the promoter and melt DNA. They
collectively perform the functions performed by sigma in bacterial transcription.
• The pre initiation complex also helps the polymerase to escape from the
promoter and embark on the elongation phase.
• The complete set of GTFs & polymerase bound together at the promoter and
paired for initiation is called as pre-initiation complex.
• Many Pol II promoters contain TATA elements [ these are 30 base pairs
upstream from the transcription start site], where pre-initiation complex
formation begins.
• The TATA elements are recognized by GTFs called TFIID.
• The components of TFIID that binds to the TATA DNA sequence is called TBP
[TATA binding factor].

27. PREINITIATION COMPLEX FORMATION
• The other subunit is TAFs that control the DNA binding activity of TBP and
some are associated for the DNA to bind at certain promoters.
• When the TBP-DNA complex is formed it provides a platform to recruit other
GTFs and polymerase itself to the promoter. The TBP extensively distorts the
TATA sequence.
• The other proteins bind to the promoter sequence in an ordered form: TFIIA,
TFIIB, TFIIF together with polymerase also binds to the TBP-DNA complex.
• At the end TFIIE & TFIIH bind to upstream of pol II resulting in the formation
of pre-initiation complex.
• Formation of this complex containing these all components is followed by
promoter melting.
• Promoter melting in eukaryotes requires hydrolysis of ATP and is mediated by
TFIIH.

28. • The large subunit of Pol II has a C- terminal domain [CTD],
which extends as a ‘tail’.
• The CTD contains a series of repeats of heptapeptide
sequence: Tyr-Ser-Pro-Thr-Ser-Pro-Ser.
• These repeat contains a site for phosphorylation buy specific
kinases including one that is a subunit of TFIIH.

29. Prokaryotic versus eukaryotic transcription
Prokaryotic transcription Eukaryotic transcription
 Prokaryotic transcription occurs in
the cytoplasm of the cell.
Eukaryotic transcription occurs in
the cell nucleus.
 RNA polymerase consists of 5
subunits.
RNA polymerase consists of 10-17
subunits.
 In this Mrna is transcribed directly
from template dna molecule.
Initially a pre-mRNA is formed and
then processed to yield a mature
mRNA.
Holoenzyme – [RNA polymerase+
sigma factor] recognizes and binds
directly to the promoter.
Promoter recognition cannot be
carried out by RNA polymerase
alone.

30. Prokaryotic transcription Eukaryotic transcription
Polycistronic type of transcription.
[encodes proteins required to carry
out reaction of a single metabolic
pathway].
Monocistronic type of transcription.
[ encoding a single polypeptide is
called as monocistronic m- RNA. One
gene at a time.
Single type of RNA polymerase
required for synthesis of all types of
RNA.
Three different types of RNA
polymerase required for synthesis of
all type of RNA.
The transcription and translation
process is coupled.
Does not occurs.

31. Fig: E. coli RNA polymerase
The complete enzyme consists of five subunits: two α, one β, one β′, and one
σ. The σ subunit is relatively weakly bound and can be dissociated from
the other four subunits, which constitute the core polymerase from-The
Cell: A Molecular Approach. 2nd edition Cooper GM.

32. • E. coli RNA polymerase, like DNA polymerase, is a complex
enzyme made up of multiple polypeptide chains.
• The intact enzyme consists of four different types of subunits,
called α, β, β′, and σ .
• The σ subunit is relatively weakly bound and can be separated
from the other subunits, yielding a core polymerase consisting
of two α, one β, and one β′ subunits.
• The core polymerase is fully capable of catalyzing the
polymerization of NTPs into RNA, indicating that σ is not
required for the basic catalytic activity of the enzyme.

33. • However, the core polymerase does not bind specifically to
the DNA sequences that signal the normal initiation
of transcription; therefore, the σ subunit is required to
identify the correct sites for transcription initiation.
• The selection of these sites is a critical element of
transcription because synthesis of a functional RNA must start
at the beginning of a gene.

34. RNA-pol of E.Coli
the holoenzyme of RNA-pol in E.Coli consists of 5
different subunits :
Subunit Molecular
Weight
No of molecules function
α
36.5 kD 2 Interacts with the regulatory
proteins and the DNA to be
transcribed.
β 151 kD 1 Catalyzes polymerization.
β' 155 Kd 1 DNA binding
σ 70 kD 1 Recognizes the promoter
region for synthesis
initiation.