Eukaryotic transcription

UNIT II: GENE EXPRESSION | TRANSCRIPTION IN EUKARYOTES Page 1 of 1
Transcription in Eukaryotes
PROKARYOTIC TRANCRIPTION EUKARYOTIC TRANSCRIPTION
Transcription takes place in the cytosol.
Because of this, the mRNA doesn't have to
travel anywhere before it can be translated
by a ribosome. In fact, a ribosome may
begin translating a mRNA before it is even
fully transcribed (while transcription is still
going on).
Transcription takes place in the nucleus.
The primary transcript also undergoes
processing steps in the nucleus in order to
become a mature mRNA. It is then
exported to the cytosol, where it can
associate with a ribosome and direct
synthesis of a polypeptide in the process of
translation.
Occurs in the cytoplasm. Occurs within the nucleus.
Transcriptional unit has one or more genes Transcriptional unit has just one gene
Coupled transcription where translation is
the rule.
Coupled transcription where translation is
not possible.
RNAs are released and processed in the
cytoplasm.
RNAs are released and processed in the
nucleus.
1 type of RNA polymerase present 3 types of RNA polymerase present
Pribnow box at -10 TATA box at -30*
RNA polymerase only requires one
additional protein (s factor). It can be said
to have a simple regulation
RNA polymerase requires several
transcription factors to initiate
transcription. It can be said to have
a complex regulation

TRANSCRIPTION IN EUKARYOTES
 Transcription is more complicated in eukaryotes than in prokaryotes because
eukaryotes possess three different classes of RNA polymerases and because of the
way in which transcripts are processed to their functional forms.
 More proteins and transcription factors are involved in eukaryotic transcription.
EUKARYOTIC RNA POLYMERASES
 In eukaryotes, three different RNA polymerases transcribe the genes for 4 types of
RNA’s.
 RNA polymerases in eukaryotes consist of multiple subunits.
 For example:
In Yeast, RNA polymerase II consists of 12 subunits and has a U - shaped structure.
The larger subunit of polymerase II has a Carboxy-Terminal Domain (CTD) which
extends as a tail. The CTD contains a series of repeats of the heptapeptide sequence;
Tyr-ser-pro-Thr-ser-pro-ser. These repeats contain sites for phosphorylation by
specific kinase. This phosphorylation has a major role in elongation phase.
TYPES OF EUKARYOTIC RNA POLYMERASES
ENZYME LOCATION FUNCTION
1 RNA polymerase I Nucleolus Synthesis of 3 subunits of the RNAs found in
the ribosomes: 28s,18s and 5.8s
2 RNA polymerase II Nucleoplasm Synthesis of messenger RNA (mRNA) and
some small nuclear RNA (snRNA)
3 RNA polymerase III Nucleoplasm Synthesis of transfer RNA (tRNA), 5s rRNA &
snRNAs which are not made by RNA pol 2
 In eukaryotes there are three RNA polymerases: I, II and III. The process includes a
proofreading mechanism.
 RNA polymerase I is located in the nucleolus, a specialized nuclear substructure in
which ribosomal RNA (rRNA) is transcribed, processed, and assembled into
ribosomes. The rRNA molecules are considered structural RNAs because they have a
cellular role but are not translated into protein. The rRNAs are components of the
ribosome and are essential to the process of translation. RNA polymerase I
synthesizes all of the rRNAs except for the 5S rRNA molecule.
 RNA polymerase II is located in the nucleus and synthesizes all protein-coding
nuclear pre-mRNAs. Eukaryotic pre-mRNAs undergo extensive processing after
transcription. RNA polymerase II is responsible for transcribing the overwhelming
majority of eukaryotic genes, including all of the protein-encoding genes which
ultimately are translated into proteins and genes for several types of regulatory RNAs,
including microRNAs (miRNAs) and long-coding RNAs (lncRNAs).
 RNA polymerase III is also located in the nucleus. This polymerase transcribes a
variety of structural RNAs that includes the 5S pre-rRNA, transfer pre-RNAs (pre-
tRNAs), and small nuclear pre-RNAs. The tRNAs have a critical role in translation:
they serve as the adaptor molecules between the mRNA template and the growing
polypeptide chain. Small nuclear RNAs have a variety of functions, including
“splicing” pre-mRNAs and regulating transcription factors.

REGULATORY SEQUENCES OF EUKARYOTIC TRANSCRIPTION
 The regulatory sequences are required for efficient transcription. All these elements
bind regulatory proteins (activators and repressors) which help or hinder
transcription from the core promoter
 The regulatory sequences include:
1. Promoters;
2. Enhancers (UAS)
3. Silencers
4. Insulators
1. PROMOTERS
 Each gene has its own promoter. There may be multiple promoter sequences in a
DNA molecule. They indicate transcription start sites.
 The promoter region lies at the start (or slightly overlaps with) the transcribed region.
 A promoter of protein coding gene encompasses about 200bp upstream of the
transcription initiation site and contains various sequence elements.
 Promoter can be divided into two regions;
a) The Core Promoter Elements
b) The Promoter Proximal Elements
a) The Core Promoter Elements b)The Promoter Proximal Elements
 These are Cis-acting sequence needed
for the transcription machinery to start
the RNA synthesis at the correct site.
 The elements are typically within 50bps
upstream of that site.
 The best characterized core promoter
elements are;
i) Initiator (Inr) :
 A short sequence element which
spans the transcription initiation
start site.
 Location between -3 to +5bp
 Py2CAPy5
 A is the first base to be transcribed
ii) TATA box or TATA element
 Goldbery-Hogness box
 located at about -25bp
 similar to -10 region of prokaryote.
 The TATA box has the seven
nucleotide consensus sequence.
 TATAAAA
 ~15% mammals
 They align RNA pol at proper site with
the help of TFIID
The initiator and TATA elements specify
where the transcription machinery
assembles and determine where
transcription will begin.
 They increase the efficiency of core
promoters.
 They are upstream from the TATA box,
in the area from 50 to 200 nucleotides
from the start site of transcription.
 The best characterized promoter
proximal elements are;
i) CAAT box:
 It is centered at about -80bp
 GGCCAATCT
ii) GC box:
 with consensus sequence
GGGCGG
 centered at about -90bp
 The promoter proximal elements
determine how and when a gene is
expressed key to this regulation are
transcription regulatory proteins called
activators, which determine the
efficiency of transcription initiation
 Typically a promoter include 2 or 3 of
these core and proximal promoter
elements
GENERIC PROMOTER

THYMIDINE PROMOTER
2. ENHANCERS
 Enhancers are elements interact with protein other than RNA polymerase and
regulate the activity of promoter.
 They are also called Upstream Activating Sites or UAS.
 They increase or enhance the efficiency of promoter
 Location varies; usually 100 or 200 bp upstream which it can be moved several 100s
or even 1000 bps upstream or downstream without alteration of their activities.
 It leads up to 200 fold increase in transcription rate of a gene.
3. SILENCERS
 These are repressing elements.
 They can function at great distance from gene (proposed by Alexander Johnson).
 They turn off the active gene.
4. INSULATORS
 Insulators block or inhibit or hinder the activity of enhancers.
TRANSCRIPTION FACTORS IN EUKARYOTES
 Unlike the prokaryotic RNA polymerase that can bind to a DNA template on its own,
eukaryotes require several other proteins, called transcription factors, to first bind to
the promoter region and then help recruit the appropriate polymerase. The completed
assembly of transcription factors and RNA polymerase bind to the promoter, forming
a transcription pre-initiation complex (PIC).
 Transcription factors recognize the promoter; RNA polymerase then binds and forms
the transcription initiation complex.
TYPES OF TRANSCRIPTION FACTORS
1. Basal Transcription factors:
 Basal transcription factors play a major role in transcription intiation.
 TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH, TFIIJ are Basal transcription factors.
2. Upstream Transcription factors:
 Upstream Transcription factors are also known as activators.
 They cover upstream region and increase efficiency of initiation.
 They recognize specific sequences and binds to proximal promoter.
 SP1, CTF family, CP1 family, C/EBP, ACF, Oct-1, Oct-2, TK

3. Regulatory Transcription factors:
 Regulatory Transcription factors bind to response elements.
 They are activated at specific time in specific tissue.
BASAL TRANSCRIPTION FACTORS
Sl
no
TRANSCRIPTION
FACTORS
STRUCTURE FUNCTION
1. TFIID
Commitment factor
Positioning factor
 Two sub parts;
1. TATA Binding Protein [TBP]
o Highly conserved
o binds to minor groove
sequence and bends DNA
~800C towards major groove
2. TBP Associating Factor [TAF]
 binds to major groove and
modifies chromatin structure
for transcription
 Multi (12) subunit complex
1 TAF + 11 TBP = 12
 Scans DNA
 Identifies promoter - TATA box
 Binds to TATA box
 Initiates transcription
2 TFIIA  Multi subunit complex
Eg: In yeast two subunits
In mammals three subunits
 Binds to promoter
 Specifically upstream to
TFIID
3 TFIIB  Single polypeptide chain  Binds to promoter
 Specifically downstream to
TFIID
 Covers -10 to +10 region
 Bridges the TATA bound
TBP and polymerase
4 TFIIF  Two subunits;
1) RAP 74: ATP dependent
helicase activity
2) RAP 38: Binds strongly to
RNA polymerase
 Required for the
recruitment of RNA
Polymerase, TFIIE and
TFIIH
5 TFIIE  Two subunits  Connects RNA polymerase
to promoter
 Downstream +30 bp
 Recruitment and regulation
of TFIIH
6 TFIIH  multi subunits (~9)  Protein kinase activity
(phosphorylates CTD tail of
RNA polymerase)
 ATPase-Helicase activity
 Promoter melting
7 TFIIJ  Undetermined  joins TFIIH and TFIIE

TRANSCRIPTION STEPS IN EUKARYOTES
 Accurate initiation of transcription of a protein coding gene involves the assembly of
RNA Polymerase II and a number of other proteins called Basal transcription factor
on the core promoters.
 Eukaryotic transcription is carried out in the nucleus of the cell and proceeds in
three sequential stages: initiation, elongation, and termination.
 Transcription involves major three steps;
1) Transcription initiation
2) Transcription elongation
3) Transcription termination
1. TRANSCRIPTION INITIATION
 Initiation is the first step of eukaryotic transcription and requires RNA polymerase
and several transcription factors to proceed.
 Eukaryotes require transcription factors to first bind to the promoter region and
then help recruit the appropriate RNA polymerase.
 The TATA box, as a core promoter element, is the binding site for a transcription
factor known as TATA-binding protein (TBP), which is a subunit of TFIID
 After TFIID binds to the TATA box via the TBP, five more transcription factors and
RNA polymerase combine around the TATA box in a series of stages to form a pre-
initiation complex.
 Transcription Factor II H (TFIIH), is involved in separating opposing strands of
double-stranded DNA to provide the RNA Polymerase access to a single-stranded
DNA template.
 The basal transcription factors and RNA Polymerase II bind to promoter elements in
a particular order in vivo to produce the complete transcription initiation complex,
also called the pre-initiation complex
 Activators and repressors, along with any associated coactivators or corepressors,
are responsible for modulating transcription rate.
 Activator proteins increase the transcription rate, and repressor proteins decrease
the transcription rate.

 RNA polymerase does not
bind to free floating DNA
 TFIID (TAF + TBP) identifies
and binds to TATA box. This
permits the association of
TFIIA and TFIIB.
 TFIIA binds upstream
 TFIIB binds downstream
 TFIIF + RNA pol II
RNA Polymerase is assorted to
promoter by TFIIF to form a
transcription complex
 TFII E
 TFIIH
 TFIIJ
orderly addition of TFIIE, TFIIH
and TFIIJ helps in the initiation
 Formation of pre-initiation
complex containing these
components is followed by
promoter melting which
requires hydrolysis of ATP.
 It is the helicase like activity
of TFIIH that stimulates
unwinding of promoter.
2. TRANSCRIPTION ELONGATION
 Once RNA polymerase II has initiated transcription, the elongation phase begins.
 RNA Polymerase II is a complex of 12 protein subunits. Specific subunits within the
protein allow RNA Polymerase II to act as its own helicase, sliding clamp, single-
stranded DNA binding protein, as well as carry out other functions.
 During transition from initiation to elongation, the RNA polymerase II enzyme sheds
initiation factors and recruits another set of factors that favor phosphorylation of the
CTD in RNA polymerase.
 Thus, phosphorylation of the CTD leads to an exchange of initiation factors for those
factors required for elongation and RNA processing.
 TFIIH unwinds ds DNA and phosphorylates C-terminal domain of largest subunit of
RNA polymerase.
 RNA polymerase moves along the template strand, synthesising an mRNA molecule,
in a same fashion as in prokaryotic transcription.
 As RNA polymerase accelerates towards downstream, the ribo-nucleotides (A, U, G &
C) are added to the template strand, enabling growth of the mRNA transcript.
 The mRNA transcript is made in a 5’ to 3’ direction, while reading the template DNA
strand in the 3' to 5' direction. The template DNA strand and RNA strand are
antiparallel.

 The movement of RNA polymerase is based on the ATP consumption.
 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 base pairs.
 Only about 8 nucleotides of newly-synthesized RNA remain base paired to the
template DNA. The rest of the RNA molecules fall off the template to allow the DNA
behind it to rewind.
3. TRANSCRIPTION TERMINATION
 In eukaryotes, actual termination of RNA polymearse activity during transcription
may take place through termination sites similar to these found in prokaryotes.
 Yet, the termination of transcription is different for the three different eukaryotic RNA
polymerases.
 These termination sites are believed to be present away from the site of the 3´ end of
mRNA are generated due to post transcription cleavage that is achieved by snurp.
 The sequence 5´-AAUAAA-3´ in mRNA 3´ end seems to be common in eukaryotic
mRNA and mutation in these sequence cause elongation of mRNA.
 The polyadenylation signals act as long terminator regions to end the transcription.
 RNA Polymerase II has no specific signals that terminate its transcription.
 In the case of protein-encoding genes, a protein complex will bind to two locations on
the growing pre-mRNA once the RNA polymerase has transcribed past the end of the
gene.
 In eukaryotes (such as humans), a primary transcript has to go through some extra
processing steps in order to become a mature mRNA. During processing, caps are
added to the ends of the RNA, and some pieces of it may be carefully removed in a
process called splicing.
TERMINATION DIAGRAM – refer class notes – dissolved transcriptosome
( separated pre-mRNA + separated RNA polymerase + transcription factors + ds DNA )

Gene expression is the process by which the instructions present in our DNA are
converted into a functional product
ADDITIONAL INFORMATION
 In eukaryotes, RNA polymerase II transcribes protein coding genes.
 The product of transcription is a precursor mRNA or pre-mRNA.
 The precursor mRNA molecule or transcript that must be modified, processed or
both to produce the mature functional mRNA molecule
 After transcription the RNA molecule is processed in a number of ways: introns are
removed and the exons are spliced together to form a mature mRNA molecule
consisting of a single protein-coding sequence.
 They are spliced and have a 5' cap and poly-A tail put on their ends.
 Capping : Addition of a methylated guanine cap confers protection to the mRNA. This
is necessary as RNA is much more unstable than DNA.
 It involves: Addition of a methylated guanine, Occurs at the 5’ end of the mRNA
transcript, Protects the mRNA from degradation
 Polyadenylation/tailing: Addition of a poly A tail confers protection to the mRNA.
This is necessary as RNA is much more unstable than DNA.
 It involves: Endonucleases* recognise a specific sequence along the mRNA transcript
and cleaves it there several adenine nucleotides are added (approximately 200) to the
transcript by the enzyme poly A polymerase, Occurs at the 3’ end, protecting the
mRNA from degradation.
 Splicing allows one genetic sequence to code for different proteins. This conserves
genetic material.
 It involves: Introns (non-coding sequences) are removed via spliceosome excision
 Exons (coding sequence) are then joined together by ligation
 It is sequence dependent and occurs within (in the middle of) the transcript
 This allows many proteins to be made from a single pre-mRNA
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Eukaryotic transcription

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