Transcription

 Formation of RNA (ribonucleic acid) over the DNA template is called

transcription.
 It is meant for taking the coded information from DNA to the site
where it is required for protein synthesis.
 In eucaryotes, transcription occurs inside the nucleus and the
transcription products move out into cytoplasm for translation.
 In prokaryotes, transcription occurs in contact with the cytoplasm as
their DNA lies in the cytoplasm.
 Principles of complementarity are used even in transcription. The
exception is that
(i) Uracil is incorporated instead of thymine opposite adenine of
template.
 (ii) Only the template or antisense strand of the DNA is transcribed.
 Both the DNA strands cannot be copied in transcription because that
will produce two types of proteins, one with correct sequence of
amino acids and the other with reverse sequence of amino acids.
 Further, if two complementary RNAs are produced simultaneously,
they would have a tendency to form double stranded RNA resulting
in non-translation of coded information into proteins.

Transcription in Prokaryotes

1. Initiation
(i) Transcription Unit
 The segment of DNA that takes part in transcription is called
transcription unit.
 It has three components (i) a promoter, (ii) the structural gene and
(iii) a terminator.

 Promoter is located upstream of structural gene. By convention it is

called 5′ end (of coding strand which is 3′ end of template strand).
 Terminator region is present downstream of structural gene at the 3′
end of coding strand which is actually 5′ end of the template strand.
 Terminator defines the end of transcription process.
 Promoter has different parts for attachment to various transcription
factors
  Structural gene in a transcription unit is flanked by the promoter and
terminator.
 Structural gene is component of that strand of DNA which has 3′→ 5′
polarity (as transcription can occur only in 5′→ 3′ direction). This
strand of DNA is called template strand or master strand or
antisense, or (-) strand.
 The other strand which has a polarity of 5′→ 3′ is called non-template
strand which does not take part in transcription. It is also called
sense or coding strand or plus (+) strand because genetic code
present in this strand is similar to genetic code (based on mRNA)
except that uracil is replaced by thymine.
(ii) Prokaryotic RNA Polymerase
 A single enzyme- DNA dependent RNA polymerase or simply RNA
polymerase synthesizes all the RNAs in prokaryotes.
 RNA polymerase of E. coli is a complex holoenzyme with five
polypeptide subunits- 2α, 1β and 1β′ and one sigma (σ) factor.
 The enzyme without sigma factor is referred to as core enzyme (α 2ββ
′).
(iii). Binding of RNA polymerase
 The binding of the enzyme RNA polymerase to DNA is the
prerequisite for the transcription to start.
 The two strands of DNA uncoil progressively from the site of
polymerase binding. Enzymes required for chain separation are
unwindases and single strand binding proteins (SSBPs).
 The region of opened-up DNA is called a transcription bubble.
 The specific region on the DNA where the enzyme binds is known as
promoter region.
 There are two base sequences on the DNA strand which the sigma
factor of RNA polymerase can recognize for initiation of transcription.
(a.) Pribnow box (TATA box)
  This consists of 6 nucleotide bases (TATAAT), located on the left
side about 10 bases away (upstream) from the starting point of
transcription.
(b.) The ‘-35’ sequence
 This is the second recognition site in the promoter region of DNA.
 It contains a base sequence TTGACA, which is located about 35
bases (upstream, hence -35) away on the left side from the starting
point of transcription.
2. Elongation
 As the RNA chain formation initiates, the sigma (σ) factor of the RNA
polymerase separates.
 RNA polymerase (core enzyme) moves along the DNA template
causing elongation of RNA chain at the rate of some 40 nucleotides
per second.
 As elongation proceeds, the DNA is continuously unwound ahead of
the core enzyme and rewound behind it.
 RNA pol catalyzes the polymerization of ribonucleoside 5′-
triphosphates (NTPs) using the DNA as template.
 The ribonucleoside triphosphates (ATP, GTP, CTP and UTP) are
added to the 3′ end, i.e. mRNA synthesis takes place in the 5′ to 3′
direction.
 They form complementary pairs, U opposite A, A opposite T, С
opposite G, and G opposite C.
 The adjacent ribonucleotides held over DNA template join by
phosphodiester bonds to form RNA chain.
 RNA synthesis stops as soon as polymerase reaches the terminator
region.
3. Termination
The process of ending transcription is called termination, and it
happens once the
polymerase transcribes a sequence of DNA known as a terminator.
(i) Rho independent or RNA based or intrinsic termination
 Rho-independent termination is controlled by specific sequences in
the DNA template strand.
 As the polymerase reaches the end of the gene being transcribed, it
encounters a region rich in C–G nucleotides.
 The mRNA folds back on itself, and the complementary C–G
nucleotides base pair with one another and form a stem-loop
structure.
 There seems to be another Adenine rich area immediately following
the inverted repetition pattern (AAAA).
 Whenever RNA polymerase advances, complementary U–A region
of the mRNA transcript forms only a weak interaction with the
template DNA.
 Such fragile Adenine-Uracil interactions break down and divide both
DNA template as well as the RNA transcript. Finally, the transcript is
released from the transcribing site.
(ii) Rho dependent Termination
 Rho is the main factor required for termination; it is a ring-shaped
hexameric protein with ATPase and helicase activities.
 Rho's ATPase is activated by Rho-RNA binding, and provides the
energy for Rho translocation along the mRNA.
 The Rho protein binds to the RNA transcript especially G-C rich sites
and moves in a 5′-3′ direction with the RNA polymerase, which
 promotes the breaking of hydrogen bonds between the DNA
template and RNA transcript.
 Rho factor separates the DNA/RNA hybrid as it approaches the
transcription bubble, releasing the transcript from the bubble. the
transcript is released by Rho's helicase activity from the bubble.
4. Duplex Formation
  After the release of primary transcript, the two strands of DNA
establish linkages amongst complementary base pairs.
 Gyrases, unwindases and SSB (single stranded binding) proteins are
released.
 Consequently, the double helical form of DNA is resumed.

Transcription in Eukaryotes

Enzymes involved in Eukaryotic Transcription

 RNA polymerase I is located in the nucleolus and transcribes the
28S, 18S, and 5.8S rRNA genes.
 RNA polymerase II is located in the nucleoplasm and transcribes
protein-coding genes, to yield pre-mRNA, and also the genes
encoding small nucleolar RNAs (snoRNAs) involved in rRNA
processing and small nuclear RNAs (snRNAs) involved in mRNA
processing, except for U6 snRNA.
 RNA polymerase III is also located in the nucleoplasm. It
transcribes the genes for tRNA, 5S rRNA, U6 snRNA, and the 7S
rRNA associated with the signal recognition particle (SRP) involved
in the translocation of proteins across the endoplasmic reticulum
membrane.
  Each of the three eukaryotic RNA polymerases contains 12 or more
subunits and so these are large complex enzymes.
Process of Eukaryotic Transcription
1. Initiation Phase
 During initiation, RNA polymerase recognizes a specific site on the
DNA, upstream from the gene that will be transcribed, called
a promoter site and then unwinds the double helical DNA.

 Most promoter sites for RNA polymerase II include a highly

conserved sequence located about -25 to -35 bp upstream of the
 start site which has the consensus TATA(A/T)A(A/T) and is called
the TATA box.
 The TATA box sequence resembles the -10 sequence in prokaryotes
(TATAAT) except that it is located further upstream.
 Transcription is also regulated by upstream control elements that lie
upstream to the TATA box.
 Some eukaryotic protein-coding genes lack a TATA box and have an
initiator element instead, centered around the transcriptional
initiation site.
 In order to initiate transcription, RNA polymerase II requires the
assistance of several other proteins or protein complexes, called
general (or basal) transcription factors, which must assemble into
 a complex on the promoter in order for RNA polymerase to bind and
start transcription.
 Eukaryotic promoters also possess enhancers (increase the rate of
transcription) and silencers (repressed the rate of transcription).
 These all have the generic name of TFII (for Transcription Factor for
RNA polymerase II)- TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH.
 TFIIA-Apple, TFIIB-Ball, TFIID-Dog, TFIIE-Elephant, TFIIF- Fan,
TFIIH-Helicopter.
 The first event in initiation is the binding of the transcription factor
TFIID protein complex to the TATA box via one its subunits called
TBP (TATA Binding Protein).
 As soon as the TFIID complex has bound, TFIIA binds and stabilizes
the TFIID-TATA box interaction. Next, TFIIB binds to TFIID.
 However, TFIIB helps in the recruitment RNA polymerase II on the
promoter and so acts as a bridging protein.
 RNA polymerase II cannot bind the promoter on its own. A
transcription factor TFIIF helps RNA polymerase II to bind the
promoter which is already complexed with TFIIF.
 This is followed by the binding of TFIIE which helps in the binding of
TFIIH.
 The final complex contains at least 40 polypeptides and is called
the transcription initiation complex.
 TFIIH is a very large complex which consists of 9 subunits. 2
subunits have ATPase activity. Using energy from ATP, it unwinds
the DNA helix, allowing transcription to begin. This results in the
formation of open complex.
 The remaining 7 subunits of TFIIH has a kinase activity which
phosphorylase the C-terminal domain (CTD) or tail of RNA
polymerase II leading to promoter escape and transcription
elongation.
 Interestingly, only RNA polymerase II that has a non-phosphorylated
CTD can initiate transcription but only an RNA polymerase II with a
phosphorylated CTD can elongate RNA.
 Those protein-coding genes that have an initiator element instead of
a TATA box appear to need another protein(s) that binds to the
initiator element.
 The other transcription factors then bind to form the transcription
initiation complex in a similar manner to that described above for
genes possessing a TATA box promoter.
The Dog (TFIID) eats the Apple (TFIIA)
And plays with the Ball (TFIIB)
He gets tired and sits in front of the Fan (TFIIF)
Then he sees the Elephant (TFIIE)
And runs away in the Helicopter (TFIIH)

2. Elongation Phase
 RNA polymerase II now starts moving along the DNA template,
synthesizing RNA, that is, the process enters the elongation phase.
 The transcription factors that helps in elongation are called
elongation factors. There are two elongation factors- TFEb and
TFIIS.
 TFEb is recruited to RNA polymerase by transcription activators.
TFEb is a kinase protein and phosphorylates serine residues in the
CTD of RNA polymerase. This phosphorylation stimulates
elongation.
 The rate at which the RNA polymerase transcribes the DNA is not
same at all DNA sequences. At some DNA sequences, the rate of
transcription is fast while at other DNA sequences it can be slow.
TFIIS increase the rate of transcription at the region where the rate
of transcription is slow. It also does not allow RNA polymerase to
pause and encourage it to move on.
  RNA synthesis occurs in the 5’ → 3’ direction.
 With the help of RNA polymerase and Mg2+, the adjacent
ribonucleotides held over DNA template join by phosphodiester
bonds to form RNA chain.
 The RNA molecule made from a protein-coding gene by RNA
polymerase II is called a primary transcript.

3. Termination Phase
 When RNA polymerase reaches end of the transcribed gene, CTD of
RNA polymerase interacts with two proteins, CStF (Cleavage
 Stimulation Factor) and CPSF (Cleavage and Polyadenylation
Specificity Factor).
 CStF cleaves the mRNA. Once the mRNA is cleaved, CStF
dissociates.
 CPSF then recruits Poly A polymerase which adds about two
hundred adenine residues at the 3’end giving rise to ploy A tail. Poly
A polymerase uses ATP for this purpose. Poly A binding protein
binds the Poly A tail and CPSF is released. Poly A binding proteins
prevents degradation of Poly A tail.
 The transcript is released, which will serve as the initial RNA prior to
further processing (the pre-mRNA).
RNA processing
 Post-transcription processing is required to convert primary
eukaryotic mRNA transcript or heterogenous nuclear RNA (hnRNA)
or pre- mRNA into functional mRNAs.
 It undergoes various processing steps to change into a mature
mRNA.
1. Cleavage
 Larger RNA precursors are cleaved to form smaller RNAs.
 Primary transcript is cleaved by ribonuclease-P (an RNA enzyme) to
form 5-7 RNA precursors.
2. Capping and Tailing
 At the 5′ end a cap (consisting of 7-methyl guanosine or 7 mG) and a
tail of poly A at the 3′ end are added.
 The cap is a chemically modified molecule of guanosine triphosphate
(GTP).

3. Splicing
 The eukaryotic primary mRNAs are made up of two types of
segments; non-coding introns and the coding exons.
 The introns are removed by a process called RNA splicing where
RNA is cut, releasing the introns and joining two adjacent exons to
produce mature mRNA.
4. Nucleotide Modifications
 They are most common in tRNA-methylation (e.g., methyl cytosine,
methyl guanosine), deamination (e.g., inosine from adenine),
dihydrouracil, pseudouracil, etc.