1. T R A N S C R I P T I O N I N
P R O K A R Y O T E S & E U K A R Y O T E S

2. I N T R O D U C T I O N
 Transcription refers to the first step of gene expression where an RNA polymer is created from a DNA
template.
 This reaction is catalysed by enzymes called RNA polymerases and the RNA polymer is antiparallel and
complementary to the DNA template.
 The stretch of DNA that codes for an RNA transcript is called a transcription unit and could contain more than
one gene.
 These RNA transcripts can either be used as messengers to drive the synthesis of proteins or be involved in a
number of different cellular processes.
 These functional or non-coding RNA could be tRNA, rRNA or direct gene regulation through RNA interference
and the formation of heterochromatin.
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3. M E C H A N I S M
O F
T R A N S C R I P T I O N
 Transcription creates a single stranded RNA
molecule from double stranded DNA.
 Therefore, only the information in one of the strands
is transferred into the nucleotide sequence of RNA.
 One strand of DNA is called the coding strand and
the other is the template strand.
 Transcription machinery interacts with the template
strand to produce an mRNA whose sequence
resembles the coding strand.
 Two different genes on the same DNA molecule can
have coding sequences on different strands.
 Transcriptional activity is particularly high in the G1
and G2 phases of the cell cycle when the cell is
either recovering from mitosis or preparing for the
dramatic events of the next cycle of cell division.
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4. The process of transcription can be broadly categorised into 3 main stages: initiation, elongation & termination.
 Transcription is catalysed by the enzyme RNA polymerase.
 It attaches to and moves along the DNA molecule until it recognises a promoter sequence, which indicates the
starting point of transcription.
 There may be multiple promoter sequences in a DNA molecule. Transcription factors are proteins that control
the rate of transcription.
 They too bind to the promoter sequences with RNA polymerase.
 Once bound to the promotor sequence, RNA polymerase unwinds a portion of the DNA double helix, exposing
the bases on each of the two DNA strands.
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INITIATION

5.  One DNA strand (template strand) is read in a 3′ to 5′ direction and so provides the template for the new
mRNA molecule.
 The other DNA strand is referred to as the coding strand.
 This is because the base sequence of the new mRNA is identical to it, except for the replacement of thiamine
bases with uracil.
 Incoming ribonucleotides are used by RNA polymerase to form the mRNA strand.
 It does this using complementary base pairing (A to U, T to A, C to G and G to C).
 RNA polymerase then catalyses the formation of phosphodiester bonds between adjacent ribonucleotides.
 Bases can only be added to the 3′ end, so the strand elongates in a 5’ to 3’ direction.
Elongation will continue until the RNA polymerase encounters a stop sequence. At this point, transcription stops
and the RNA polymerase releases the DNA template.
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ELONGATION
TERMINATION

6. PROKARYOTIC PROMOTERS & INITIATION OF TRANSCRIPTION
o The nucleotide pair in the DNA double helix that corresponds to the site from which the first 5′ mRNA
nucleotide is transcribed is called the +1 site, or the initiation site.
o Nucleotides preceding the initiation site are given negative numbers and are designated upstream.
o Conversely, nucleotides following the initiation site are denoted with “+” numbering and are called
downstream nucleotides.
o A promoter is a DNA sequence onto which the transcription machinery binds and initiates transcription. In
most cases, promoters exist upstream of the genes they regulate.
o The specific sequence of a promoter is very important because it determines whether the corresponding
gene is transcribed all the time, some of the time, or infrequently.
o Although promoters vary among prokaryotic genomes, a few elements are conserved.
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7. o At the -10 and -35 regions upstream of the initiation site, there are two promoter consensus sequences, or
regions that are similar across all promoters and across various bacterial species.
o The -10 consensus sequence, called the -10 region, is TATAAT. The -35 sequence, TTGACA, is recognized
and bound by σ.
o Once this interaction is made, the subunits of the core enzyme bind to the site. The A–T-rich -10 region
facilitates unwinding of the DNA template; several phosphodiester bonds are made.
o The transcription initiation phase ends with the production of abortive transcripts, which are polymers of
approximately 10 nucleotides that are made and released.
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8. ELONGATION IN PROKARYOTES
o The transcription elongation phase begins with the release of the σ subunit from the polymerase.
o The dissociation of σ allows the core RNA polymerase enzyme to proceed along the DNA template,
synthesizing mRNA in the 5′ to 3′ direction at a rate of approximately 40 nucleotides per second.
o As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it.
o Since the base pairing between DNA and RNA is not stable enough to maintain the stability of the mRNA
synthesis components, RNA polymerase acts as a stable linker between the DNA template and the nascent
RNA strands to ensure that elongation is not interrupted prematurely.
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9. TERMINATION IN PROKARYOTES
o Once a gene is transcribed, the prokaryotic polymerase needs to be instructed to dissociate from the DNA
template and liberate the newly-made mRNA.
o Depending on the gene being transcribed, there are two kinds of termination signals: one is protein-based and
the other is RNA-based.
o Rho-dependent termination is controlled by the rho protein, which tracks along behind the polymerase on the
growing mRNA chain.
o Near the end of the gene, the polymerase encounters a run of G nucleotides on the DNA template and it stalls.
o As a result, the rho protein collides with the polymerase. The interaction with rho releases the mRNA from the
transcription bubble.
o Rho-independent termination is controlled by specific sequences in the DNA template strand.
o As the polymerase nears the end of the gene being transcribed, it encounters a region rich in C-G nucleotides.
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10. o The mRNA folds back on itself, and the complementary C–G nucleotides bind together. The result is a
stable hairpin that causes the polymerase to stall as soon as it begins to transcribe a region rich in A-T
nucleotides.
o The complementary U-A region of the mRNA transcript forms only a weak interaction with the template
DNA.
o This, coupled with the stalled polymerase, induces enough instability for the core enzyme to break away
and liberate the new mRNA transcript.
o Upon termination, the process of transcription is complete.
o By the time termination occurs, the prokaryotic transcript would already have been used to begin synthesis
of numerous copies of the encoded protein because these processes can occur concurrently in the
cytoplasm.
o The unification of transcription, translation, and even mRNA degradation is possible because all of these
processes occur in the same 5′ to 3′ direction and because there is no membranous compartmentalization
in the prokaryotic cell.
o In contrast, the presence of a nucleus in eukaryotic cells prevents simultaneous transcription and
translation.
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11. INITIATION OF TRANSCRIPTION 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).
 The most-extensively studied core promoter element in eukaryotes is a short DNA sequence known as a
TATA box, found 25-30 base pairs upstream from the start site of transcription.
 Only about 10-15% of mammalian genes contain TATA boxes, while the rest contain other core promoter
elements, but the mechanisms by which transcription is initiated at promoters with TATA boxes is well
characterized.
 The TATA box, as a core promoter element, is the binding site for a transcription factor known as TATA-
binding protein (TBP), which is itself a subunit of another transcription factor:
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12.  Transcription Factor II D (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.
 One transcription factor, 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.
 However, only a low, or basal, rate of transcription is driven by the pre-
initiation complex alone.
 Other proteins known as 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.
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13. TRANSCRIPTION THROUGH NUCLEOSOMES
 Following the formation of the pre-initiation complex, the polymerase is released from the other transcription
factors, and elongation is allowed to proceed with the polymerase synthesizing RNA in the 5′ to 3′ direction.
 RNA Polymerase II (RNAPII) transcribes the major share of eukaryotic genes, so this section will mainly focus
on how this specific polymerase accomplishes elongation and termination.
 Although the enzymatic process of elongation is essentially the same in eukaryotes and prokaryotes, the
eukaryotic DNA template is more complex.
 When eukaryotic cells are not dividing, their genes exist as a diffuse, but still extensively packaged and
compacted mass of DNA and proteins called chromatin.
 The DNA is tightly packaged around charged histone proteins at repeated intervals.
 These DNA histone complexes, collectively called nucleosomes, are regularly spaced and include 146
nucleotides of DNA wound twice around the eight histones in a nucleosome like thread around a spool.
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14. THE THREE EUKARYOTIC RNA POLYMERASES (RNAPs)
 The features of eukaryotic mRNA synthesis are markedly more complex
those of prokaryotes.
 Instead of a single polymerase comprising five subunits, the eukaryotes
have three polymerases that are each made up of 10 subunits or more.
 Each eukaryotic polymerase also requires a distinct set of transcription
factors to bring it to the DNA template.
 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.
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15.  RNA polymerase II is located in the nucleus and synthesizes all protein-coding nuclear pre-mRNAs.
 Eukaryotic pre-mRNAs undergo extensive processing after transcription, but before translation.
 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. Not all miRNAs are transcribed by RNA Polymerase II, RNA Polymerase III
transcribes some of them.
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16. ELONGATION
 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.
 Consequently, RNA Polymerase II does not need as many accessory proteins to catalyse the synthesis of
new RNA strands during transcription elongation as DNA Polymerase does to catalyse the synthesis of new
DNA strands during replication elongation.
 However, RNA Polymerase II does need a large collection of accessory proteins to initiate transcription at
gene promoters, but once the double-stranded DNA in the transcription start region has been unwound, the
RNA Polymerase II has been positioned at the +1 initiation nucleotide.
 It has started catalysing new RNA strand synthesis, RNA Polymerase II clears or “escapes” the promoter
region and leaves most of the transcription initiation proteins behind.
 All RNA Polymerases travel along the template DNA strand in the 3′ to 5′ direction and catalyse the
synthesis of new RNA strands in the 5′ to 3′ direction, adding new nucleotides to the 3′ end of the growing
RNA strand.
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17.  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 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 time.
 Whichever RNA nucleotide is capable of base pairing 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 catalysed, the RNA Polymerase moves to the next DNA nucleotide on the
template below it. This process continues until transcription termination occurs.
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18. TERMINATION
 The termination of transcription is different for the three different eukaryotic RNA polymerases.
 The ribosomal rRNA genes transcribed by RNA Polymerase I contain a specific sequence of base pairs 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.
 The protein-encoding, structural RNA, and regulatory RNA genes transcribed by RNA Polymerase 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.
 This cleavage site is considered the “end” of the gene. 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.
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19.  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.
 In the case of protein-encoding genes, the cleavage site which determines the
“end” of the emerging pre-mRNA occurs between an upstream AAUAAA sequence
and a downstream GU-rich sequence separated by about 40-60 nucleotides in the
emerging RNA.
 Once both of these sequences have been transcribed, a protein called CPSF in
humans binds the AAUAAA sequence and a protein called CstF in humans binds
the GU-rich sequence.
 These two proteins form the base of a complicated protein complex that forms in
this region before CPSF cleaves the nascent pre-mRNA at a site 10-30 nucleotides
downstream from the AAUAAA site.
 The Poly(A) Polymerase enzyme which catalyses the addition of a 3′ poly-A tail on
the pre-mRNA is part of the complex that forms with CPSF and CstF.
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20. D I F F E R E N C E
PROKARYOTIC TRANSCRIPTION EUKARYOTIC TRANSCRIPTION
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 Transcription and translation are continuous process
and occurs simultaneously in the cytoplasm.
 They are two separate processes, transcription occurs
in the nucleus whereas translation occurs in the
cytoplasm.
 Transcription initiation machinery is simple since DNA
is not associated with any histone proteins.
 Transcription initiation machinery is very complex since
the genetic material is associated with proteins.
 Only one type of RNA polymerase enzyme, which
synthesize all types of RNA in the cell (mRNA, rRNA &
tRNA)
 Three types of RNA polymerase in the cell. RNA
polymerase I for rRNA synthesis, RNA polymerase II
for mRNA synthesis. RNA polymerase III for tRNA and
5S rRNA synthesis.
 RNA polymerase with 5 subunits, two α subunits, one β
subunit, one β’ subunit, one ω subunit.
 RNA polymerase I with 14 subunits, RNA polymerase II
with 10–12 subunits, RNA polymerase III with 12
subunits.

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 σ factor present, which is essential for transcription
initiation.
 σ factor absent and it is not required for transcription
initiation. Initiation of transcription is facilitated by
initiation factors.
 RNA polymerase can recognise and bind to the
promoter region with the help of σ factor.
 RNA polymerase cannot recognise the promoter region
directly unless the promoter is pre-occupied by
transcription initiation factors.
 Promoter region always located upstream to the start
site.
 Promoter region usually located upstream to the start
site, but rarely as in the case of RNA polymerase III,
promoter is located downstream to start site.
 Promoter region contain Pribnow box at -10 positions.
TATA box and CAT box are absent in the promoter
region of the prokaryotes.
 Promoter region contains; TATA box located 35-25
upstream; CAT box located ̴ 70 nucleotide upstream;
GC box located ̴ 110 nucleotide upstream. Pribnow box
absent in eukaryotes.
 Termination of transcription is done either by ρ (Rho)
dependent mechanisms or ρ (Rho) independent
mechanisms.
 A termination mechanism of transcription is not
completely known. It may be direct by the poly A signal
or by the presence of termination sequence in the DNA.

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 Usually there is no post transcriptional modification of
the primary transcript.
 Primary transcript undergo post transcriptional
modifications (RNA editing)
 RNA capping absent, mRNA is devoid of 5’ guanosine
cap.
 RNA capping present, capping occurs at the 5’ position
of mRNA.
 Poly A tailing of mRNA is absent.  Mature mRNA with a poly A tail at the 3’ position. Poly A
tail is added enzymatically without the complementary
strand.
 Introns absent in the mRNA.  Introns present in the primary transcript.
 Splicing of mRNA absent since introns are absent.  Splicing present, introns in the primary transcript are
removed and exons are rejoined by a variety of splicing
mechanisms.
 Genes usually polycistronic and hence single transcript
may contain sequence for many polypeptides.
 Genes are monocistronic thus single transcript code for
only one polypeptide.
 SD – sequence (Shine – Dalgarno sequence) present
about 8 nucleotide upstream of start codon in the
mRNA, SD sequence act as the ribosome binding site.
 SD sequence is absent in mRNA of prokaryotes.

24. F U N C T I O N S
 Life on earth is said to have begun from self-replicating RNA since it is the only class of molecules capable of
both catalysis and carrying genetic information.
 With evolution, proteins took over catalysis because they are capable of a greater variety of sequences and
structures.
 Additionally, the bonds on the sugar phosphate backbone of RNA are vulnerable to even mild changes in pH
and can undergo alkaline hydrolysis.
 Therefore, DNA emerged as the preferred molecule for carrying genetic information since it is more stable and
resistant to degradation.
 Transcription maintains the link between these two molecules and allows cells to use a stable nucleic acid as
the genetic material while retaining most of their protein synthesis machinery.
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25. C O N C L U S I O N
 Separating DNA from the site for protein
synthesis also protects genetic material from
the biochemical and biophysical stresses of
complex, multi-layered processes.
 Small errors in the RNA transcript can be
overcome since the RNA molecule has a short
half-life, but changes to the DNA become
heritable mutations.
 In addition, transcription adds another layer for
intricate gene regulation, especially
in species with large genomes that require
minute adjustments in metabolism.
 In eukaryotes, transcription also plays an
important role in transferring the information
from DNA to the cytoplasm because the nuclear
pore is too small to allow ribosomes, proteins or
chromosomes to pass through.
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