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Paper: Molecular Biology
Lesson: Transcription
Author Name: Hardeep Kaur
College/ Department: Ramjas College,
Department of Zoology , University of Delhi
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Title- Transcription
1. Mechanism of Transcription in Prokaryotes
Transcription is a process involving synthesis of RNA on DNA template in such a way that the
nitrogenous base sequence of RNA is similar to one of the two strands of parent DNA. This DNA
strand with similar nitrogenous base sequence to transcribed RNA is called the coding strand and
the other strand of the DNA is called the template strand (Fig 1). Transcription is said to be
asymmetrical as only one strand is used as template for transcription. The difference in the base
composition of transcribed RNA and coding strand of DNA is presence of uracil in place of
thymine. The enzyme RNA polymerase is involved in RNA synthesis, which catalyzes the
polymerization of ribonucleoside 5′-triphosphates (NTPs).The enzyme, catalyzes the growth of
RNA chains always in the 5′ to 3′ direction. In prokaryotes, the genetic material is not enclosed
within a nuclear envelope therefore; the newly transcribed mRNA is available for translation
simultaneously. The process of transcription and translation are hence coupled in prokaryotes.
Fig 1. Coding and Template strand during transcription in prokaryotic cell
1.1 Prokaryotic (E.coli) RNA polymerase
The RNA polymerase of Escherichia coli uses the base sequence of one of the DNA strands. In
prokaryotes, a single RNA polymerase is responsible for transcription of mRNA, tRNA and
rRNA. RNA polymerase is one of the largest enzymes in the cell and consists of at least five
subunits (Fig 2). These subunits areInstitute of Lifelong Learning, University of Delhi
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a) Alpha (α)
b) Beta (β)
c) Beta prime (β´)
d) Omega (ω)
e) Sigma (σ)
Fig 2. RNA polymerase: Core Enzyme + Sigma factor = Holoenzyme
The bacterial RNA polymerase (RNAP) consists of a core enzyme which has two identical α
subunits and one each of β, β´ and ω (~400 kDa).The core enzyme can bind with σ factor to form
complete RNA polymerase, which is called the ‘holoenzyme’. The holoenzyme therefore has 6
subunits: β'βαI and αIIωσ (~450 kDa).The two largest subunits (β and β´) of the core enzyme are
involved in catalysis while α and ω helps in polymerase assembly and regulation. The sigma (σ)
helps in recognition and selection of start signal (initiation sites of transcription) on DNA
molecule. In the absence of the σ factor, initiation of RNA synthesis takes place in a random
manner. The magnesium ion located at the enzyme active site plays a critical role in the process
(Fig 3). This divalent ion occupies a specific site in the enzyme that leads to activation of the
enzyme. Once RNA synthesis is initiated and the RNA is about 8-9 bases long, σ factor
dissociates. The amount of σ factor is only 30% of the core enzyme in a cell. Therefore, at any
given time, only one-third of the polymerase can exist as holoenzymes.
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Fig 3. RNA Polymerase from Thermus aquaticus pictured during elongation.
(The magnesium ion located at the enzyme active site.)
The most common E.coli sigma factor is σ70 (molecular mass of 70KDa) though some other
sigma factors (σ32, σ41.5, σ54, σ28) are also known to occur which operate under adverse conditions
like high temperature, nutrient starvation, nitrogen deficiency etc.
1.2 E.coli σ70 Promoter
In transcription, by convention upstream/downstream refers to the position of DNA sequence(s)
with respect to specific transcription unit. RNA polymerase moves from 5’ to 3’ direction of
coding stand. The sequence located 5’ to a particular sequence/transcription unit is called
upstream sequence while the one located after a particular sequence relative to direction of
transcription is the downstream sequence.
RNA polymerase binds to specific initiation sites which are located upstream from sequences to
be transcribed. These are called promoters. The start site of transcription is generally assigned as
position +1. In accordance with this, promoter sequences are assigned a negative number,
reflecting the distance upstream from the start of transcription. The σ70 promoter consists of a
sequence of about 40 to 60 bp. Two 6bp sequences at positions -10 and -35 are found to be of
prime importance for effective promoter functions in E. coli (Fig.4).
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-10 Sequences: Many E. coli genes have a 6 bp sequence in its promoter which is highly
conserved sequence of σ70 promoter. This sequence is centered at about -10 position. This
sequence is called the Pribnow box as it was first discovered by Pribnow in 1975. It has a
consensus sequence of “TATAAT”. The first two bases (TA) and the final T are the highly
conserved among promoter of other genes in E. coli. This is the sequence at which DNA
unwinding is initiated by the enzyme helicase. Helicase is the enzyme involved in separating the
strands of DNA double helix using ATP as the energy source. It is an enzyme with high
specificity.
-35 Sequence: In most prokaryotic promoters, upstream regions around position -35 also have a
conserved hexamer sequence of “TTGACA” which is most conserved.
TTGACA………16-18bp……….TATAAT…….5-8bp………..CG/AT
-35
-10
+1
Transcription Start Site- The transcription start site is a purine often adenine in 90% of genes in
prokaryotes.
Fig 4. Prokaryotic Promoter Sequence
The functions of different promoter regions (Fig 4.) are as followsa) The -35 sequence is a region that enhances recognition and interaction with the polymerase σ
factor.
b) The -10 region is involved in DNA unwinding.
c) Transcription initiates at transcription start site.
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The sequence of the first 30 bases that has to be transcribed also influences transcription.
The differences in the consensus sequence of the promotor of different genes result in difference
in the rate of transcription. The promoter may bind to accessory activating factor in order to
augment polymerase binding and initiation of transcription. For example lac promoter Plac
requires cAMP receptor protein (CRP) to bind to a site on the DNA close to the promoter
sequence in order to enhance polymerase binding and initiation of transcription.
1.3 Initiation, Elongation and Termination of Prokaryotic Transcription
The process of Prokaryotic DNA transcription has been summarized in Fig 5.
Fig 5.Process of Prokaryotic DNA Transcription
RNA synthesis by RNA polymerase proceeds in the following steps1.3.1 Initiation
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1)
Binding of RNA polymerase with the Promoter- The σ factor help in recognition of
promotor site of a gene to be transcribed. The holoenzyme then binds to the promoter site.
This leads to the formation of a “closed promoter complex” in which DNA remains double
helical.
The RNA polymerase core enzyme α2ββ´ω has a general nonspecific affinity for DNA
which leads to loose binding of the enzyme to the DNA. However with the attachment of σ
factor to the core enzyme, the non-specific binding becomes highly specific. In addition σ
factor enhances holoenzyme binding to the correct promoter binding site and hence increases
its specificity. The holoenzyme further slides along the DNA and finds the double-stranded
-35 and -10 DNA sequences to form a closed complex with DNA which remains double
stranded at this stage.
2)
Unwinding of DNA-The closed complex isomerizes which causes unwinding and separation
of two DNA strands to form an open complex; this process is called tight binding.
3)
Initiation of RNA synthesis- Regulation of transcription occurs at initiation. RNA synthesis
does not require a primer. Alternatively, transcription starts de novo at specific sites at the
beginning of genes. The chain is started with a GTP or ATP. After unwinding, only one of
the strands is used for transcription. Initially upto 9 nucleotides are added to newly
synthesized RNA without movement of the enzyme, RNA polymerase. During this, at every
step, there is a possibility for the release of this small RNA chain of short 2-9 bases. This
process is described as ‘abortive initiation’. The minimum time for promoter clearance is 1-2
seconds, which is a long event as compared to the other stages of transcription.
1.3.2 Elongation
After successful initiation, the enzyme releases the σ factor and forms a ‘Ternary Elongation
Complex’ (three components) of polymerase-DNA-nascent RNA, causing the polymerase to
progress along the DNA (promoter clearance). Reinitiation of transcription from the promoter by
another RNA polymerase holoenzyme is possible at this time.
The region of unwound DNA which is about 17 bp long is called the ‘Transcription bubble’ and
appears to move along the DNA with the polymerase. The polymerase seems to unwind DNA in
front of the transcription bubble and rewinds DNA at its rear. It moves at an average rate of 40
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bases per second at 37ºC. However in some phages (T3 and T7) RNA polymerase synthesizes
RNA very rapidly (~200 bases/second at 37ºC). Another important characteristic of RNA
polymerase is its fidelity. The Fidelity of RNA polymerase can be defined as its ability to
accurately transcribe the DNA template and this is ensured by proofreading mechanisms which
removes the wrongly incorporated base.
1.3.3 Termination1. Rho-independent termination- The RNA polymerase continues transcription until it reaches a
terminator sequence (stop signal) at the end of the transcription unit. This stop signal is generally
a hairpin loop in newly transcribe RNA followed by poly U (Fig 6). This is a GC rich region, in
which the RNA transcript is self-pairing due to complementary base pairs. RNA therefore can
form a stable hairpin structure. The hairpin is often followed by a sequence of four or more U
residues. After synthesis of the hairpin in the transcript, the polymerase pauses. The stretch of U
residues after hairpin in the transcript base pair weakly with the A residues present in the
antisense DNA strand. This weak binding leads to removal of the RNA from the transcription
complex. The RNA is hence released. The non-base-paired antisense strand of DNA then reanneals with the sense DNA strand. This leads to the dissociation of the core enzyme from the
DNA.
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Fig 6. Rho-independent termination- RNA hairpin structure
Source: https://adapaproject.org/bbk (creative commons)
2 Rho-dependent termination: Besides the use of hair pin structure dependent termination,
the other terminator sites use an accessory factor, the rho (ρ) to mediate termination of
transcription. Rho (ρ) is a hexameric protein. It hydrolyzes ATP in the presence of singlestranded RNA. Rho (ρ) binds to a stretch of 72 nucleotides in RNA. This protein then moves
along the nascent RNA towards the transcription complex where it enables the RNA
polymerase to terminate at rho-dependent transcriptional terminators.
As was the case in rho-independent termination, these signals are recognized in newly
synthesized RNA rather than in template DNA.
Interesting FactsIn 1959, Severo Ochoa won the Nobel Prize in Physiology/Medicine. Ochoa developed a process
for synthesizing RNA in vitro using polynucleotide phosphorylase.
In 2006, Roger D. Kornberg won the Nobel Prize in Chemistry. Kornberg was awarded for his
studies of the molecular basis of eukaryotic transcription.
2. Mechanism of Transcription in Eukaryotes
Eukaryotes have multiple types of nuclear RNAP. Each RNAP is responsible for synthesis of a
different subset of RNA.
2.1 RNA Polymerases in Eukaryotes

TYPE
PRODUCT
LOCATION
I
rRNA
Nucleolus
II
mRNA, SnRNA. SnoRNA
Nucleoplasm
III
5S rRNA, tRNA, U6SnRNA
Nucleoplasm
RNA polymerase I synthesizes a pre-rRNA 45S (35S in yeast). This pre-rRNA is
chemically processed to form a 28S, an 18S and a 5.8S rRNAs which form the major
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RNA constituents of the ribosome. RNA polymerase I is located in the nucleoli. It is
insensitive to α–amanitin (an amatoxin obtained from Amanita genus of mushrooms).

RNA polymerase II synthesizes pre mRNAs, most snRNA and micro RNAs. The premRNAs is processed after synthesis by formation of a cap at the 5’ end of mRNA and the
addition of poly (A) tail at the 3’ end. The introns are removed and exons, the coding
sequences of the RNA are joined during the process of RNA splicing. A range of
transcription factors are required for binding of RNA polymerase to promoters. RNA
polymerase II is located in the nucleoplasm. It is very sensitive to α–amanitin.

RNA polymerase III synthesizes tRNAs, rRNA 5S and other small RNAs that are found in
the nucleus and cytosol. The enzyme is located in the nucleoplasm. It is moderately
sensitive to α–amanitin.

RNA polymerase IV synthesizes siRNA

RNA polymerase V synthesizes RNAs involved in siRNA-directed heterochromatin.
Both RNA Pol IV and V are found exclusively in plants
All the polymerases are large enzymes (50,000 daltons), each containing 12 or more subunits.
They contain subunits which have homology to E.coli core RNA polymerase α2ββ´ subunits. The
largest two subunits are similar to each other and to the β and β´ subunits of E. coli RNA
polymerase. Other subunits in each enzyme have homology to the α-subunit of the E.coli enzyme.
Five additional subunits are common to all three polymerases. There are other subunits which are
polymerase specific.
Although both DNA and RNA polymerase carry out template-dependent nucleotide
polymerization, x-ray crystallographic studies of both these enzymes however revealed that,
other than containing a critical Mg2+ ion at the catalytic site, these enzymes are virtually
unrelated to each other.
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As in the case of the prokaryotic RNA polymerase enzyme, eukaryotic enzymes catalyze
transcription in a 5’ to 3’direction and synthesize RNA complementary to the antisense template
strand. Likewise, the eukaryotic RNA polymerase does not require a primer for initiation of
transcription. However, the eukaryotic RNA polymerase requires the presence of additional
initiation proteins before it can to bind to promoters and start transcription.

DNA-directed RNA polymerase II subunit RPB1–RPB1 subunit, largest subunit of
RNA polymerase II, is encoded by the POLR2A gene. It contains a carboxy-terminal
domain (CTD) composed of up to 52 heptapeptide repeats (YSPTSPS) that are essential
for the activity and vitality of the polymerase. In combination with several other
polymerase subunits, it forms the DNA binding domain of the polymerase, a groove in
which the DNA template is transcribed into RNA. RPB1 binds strongly with RPB8. The
CTD sequence of RPB1 may be phosphorylated at serines and some tyrosines. In vitro
studies have shown that CTD is unphosphorylated initially but gets phosphorylated once
transcription elongation starts and polymerase moves away from the promoter.

RPB2–RPB2 is the second-largest subunit, encoded by POLR2B. This subunit along with
at least two other polymerase subunits forms a structure
within the polymerase. This structure maintains contact
in the active site of the enzyme between the DNA
template and the newly synthesized RNA.

RPB3–RPB3 is the third-largest subunit of RNA Pol II,
which is encoded by POLR2C. It is hereodimeric with
another polymerase subunit, POLR2J forming a core
subassembly. It also interacts strongly with RPB1-5, 7,
10-12.

RPB4–This subunit is encoded by the POLR2D gene
and is the fourth-largest subunit. This subunit may have
a stress protective role.

Do you know…?
Nuclear RNA Polymerases
IV and V (Pol IV and V) are
found exclusively in plants.
They are specialized forms
of RNA polymerase II which
is an essential enzyme of all
eukaryotic organisms. These
enzymes evolved from Pol II
over the past several million
years (12 subunits in both
Pol IV and Pol V
correspond exactly to the 12
subunits of Pol II)
RPB5–This subunit is encoded by the POLR2E gene in
humans. Two molecules of this subunit are present in
each RNA polymerase II. It strongly interacts with
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RPB1, RPB3, and RPB6.

RPB6 - This subunit is encoded by the POLR2F gene and forms a structure with at least
two other subunits. This structure stabilizes the transcribing polymerase on the DNA
template.

RPB7- This subunit is encoded by POLR2G. It may play a role in regulating polymerase
function. RPB7 interacts strongly with RPB1 and RPB5.

RPB8 -This subunit is encoded by the POLR2H gene. It interacts with subunits RPB1-3,
5, and 7.

RPB9 - This subunit is encoded by the POLR2I gene. The groove in which the DNA
template is transcribed into RNA is composed of RPB9 and RPB1.

RPB10 - This subunit is encoded by the POLR2L. It interacts with RPB1-3 and 5, and
strongly with RPB3.

RPB11 - The RPB11 subunit is itself composed of three subunits in humans: (RPB11-aencoded by POLR2J), (RPB11-b- encoded by POLR2J2) and RPB11-c-POLR2J3).

RPB12 - This subunit is encoded by the POLR2K. It also interacts with RPB3.
2.2 Eukaryotic Promoters
Eukaryotic promoters are varied. They can be about 100–1000 base pairs long, located upstream
from the transcription initiation site. They can have URE or upstream regulatory elements
located several kilobases away from the transcriptional start site (enhancers).
Eukaryotic promoters are mainly composed of three main regions-the TATA box, the CAAT box
and the enhancer (Fig.7). The conserved sequence TATAA/TAT/A, also called HognessGoldberg box, occurs 20-30 nucleotides before the start of transcription of the vast majority of
protein coding genes. In prokaryotes, there is a similar AT-rich region but it is located slightly
closer (-10) to the transcription site. The TATA box is important for transcription. Its main
function seems to be to position RNA polymerase precisely to start the process of transcription.
The TATA box is bound by specific protein or transcription factor. Many genes have the
conserved sequence CAAT between positions -70 and -80. The importance of this region can be
determined by the fact that when this sequence is removed by deletion, the rate of transcription is
drastically reduced, emphasizing that this region probably determines the efficiency with which a
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gene is expressed. The CAAT box is also bound by a special transcription protein factor in living
cells.
Fig.7. Eukaryotic Promoter
Upstream Regulatory Elements (UREs)–These are located within 100-200 bp upstream from the
promoters. These elements greatly increase the activities of promoters. Two common examples
are SP1 box and CCAAT box. Promoters may have only one, both or multiple copies of these
sequences.
Enhancers: Enhancer is a segment of DNA that can increase expression of some gene, regardless
of their orientation. They can work at long distance of more than 1kb from a gene. Enhancer
sequence was first observed in the genome of the DNA virus SV 40. Enhancer sequences are
characteristically 100-200 bp long and contain multiple sequence elements which contribute to
their activity. They may be in all cells i.e. ubiquitous or cell type-specific.
2.3 Transcription Factors and Pre-initiation Complex Formation
Eukaryotic promoter sequences usually bind with certain proteins called transcription factors.
These factors are involved in the formation of the preinitiation transcriptional complex (Fig 8).
They function either by promoting (as an activator), or blocking (as a repressor) the recruitment
of RNA polymerase to specific genes. The number of transcription factors found within an
organism increases with genome size, and larger genomes tend to have more transcription factors
per gene. Many transcription factors have been identified and purified, which are required for
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transcription initiation from RNA Pol II promoter sequences in vitro. They have been named
transcription factor TFIIA, TFIIB etc.
Subunits in the TFIID complex
include:
 TBP (TATA binding protein)
 TAF1 (TAFII250)*
 TAF2 (CIF150)
 TAF3 (TAFII140)
 TAF4 (TAFII130/135)
 TAF4B (TAFII105)
 TAF5 (TAFII100)
 TAF6 (TAFII70/80)
 TAF7 (TAFII55)
 TAF8 (TAFII43)
 TAF9 (TAFII31/32)
 TAF9B (TAFII31L)
 TAF10 (TAFII30)
 TAF11 (TAFII28)
 TAF12 (TAFII20/15)
 TAF13 (TAFII18)
 TAF15 (TAFII68)
*TFIID- 250 kDa subunit (TAFII-250) or
TAF-TBP-associated factor 250 kDa (p250)
Fig 8. Transcription in Eukaryotes-Preinitiation Complex
(Source-www.slideplayer.com)
For video/ animation: http://youtu.be/WsofH466lqk
TFIID: TFIID is one of several transcription factors that make
up the RNA polymerase II preinitiation complex. Before the
start of transcription, the transcription Factor II D (TFIID)
complex binds to the TATA box in the promoter of the gene.
TFIID is a multiprotein complex in which one protein, TATAbinding protein (TBP) binds the TATA box. The complex also
contains other polypeptides known as TBP-associated factors
(TAFIIs).
TBP: TBP is a monomeric protein. TBP, along with a variety of TBP-associated factors (TAFIIs),
make up the TFIID, a general transcription factor that in turn makes up part of the RNA
polymerase II preinitiation complex. All eukaryotic TBPs have highly conserved C-terminal
domains of 180 residues. TBP seems to have a saddle-like structure that interacts with DNA in
the minor groove of DNA by beta sheets so that the inside of the saddle binds to DNA at the
TATA box and the outside surface of the protein is available for interactions with other protein
factors. TBP is involved in DNA denaturation (double strand separation) by bending the DNA by
80° (the AT-rich sequence to which it binds facilitates easy melting).
TFIIA: TFIIA interacts with the TBP subunit of TFIID and helps in the binding of TBP to
TATA-box containing promoter DNA. TFIIA has 3 subunits. Binding of TFIIA to TFIID
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actually prevents binding of certain inhibitors like DR1 and DR2 to TFIID thus allowing the
initiation assembly process to continue.
TFIIB: Transcription factor TFIIB confines to the nucleus where it forms a complex (the DAB
complex-synonym for TFIID-TFIIA-TFIIB complex) with transcription factors TFIID and
TFIIA. The protein serves as a bridge between transcription factor IID and RNA polymerase II.
RNA polymerase II is escorted to the promoter by TFIIF to form transcription complex.
Other Factors: After RNA polymerase binding, three other transcription factors TFIIE, TFIIH
and TFIIJ also get associated with the complex. These factors are essential for the transcription
in vitro. TFIIH is a large complex and is made up of at least 5 subunits. Activation of TFIIH
leads to phosphorylation of CTD (carboxyl terminal domain) of RNA pol II.
2.4 Initiation, Elongation and Termination of Eukaryotic Transcription
Eukaryotic RNA synthesis starts and generally ends at precise sites on the DNA (promoter and
terminator sequences).
2.4.1 Initiation
The transcription initiation involves the formation of Pre-initiation complex as already discussed.
The promoters of RNA polymerase II which lack TATA box have the initiation element
overlapping their start site. In such cases TBP is guided to such promoters by a DNA binding
protein. Further, other transcription factors are then engaged by TBP and the following events
take place as in case of TATA box promoters.
2.4.2 Elongation
Eukaryotes RNA polymerase II plays a major role in transcription. After the formation of preinitiation complex, polymerase gets free of the transcription factors and further elongation takes
place in 5’ to 3’ direction. Certain subunits(out of 12 subunits) act as helicase causing unwinding
of the double stranded DNA, while others carry out different functions. Subsequently RNA
polymerase does not need any accessory proteins for further elongation. RNA Polymerases
unwind the double stranded DNA ahead of them and allow the unwound DNA behind them to
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rewind. Only about 8 nucleotides of newly-synthesized RNA remain base paired to the template
DNA. The RNA polymerase travels along the template DNA one nucleotide at a time. This
process continues until transcription termination occurs.
The RNA precursor chains which get associated with proteins to form ribonucleoprotein particles
(RNPs), present a classic Christmas tree like configuration. The trunk of each "Christmas tree" (a
transcription unit) represents a DNA molecule; the tree branches (granular strings attached to the
DNA) are RNA molecules that have been transcribed from the DNA. As the transcription
apparatus moves down the DNA, transcribing more of the template, the RNA molecules become
longer and longer.
Processing of initial transcripts into smaller RNAs generally starts before the RNA chains are
completed. A very small fraction only of chromatin is transcribed at a given time.
During transcription, the RNA polymerase molecules are packed very close together, with one
polymerase every 200 bp of DNA or less. (The elongation rate of RNA synthesis is 20-30
nucleotides per second therefore a maximally expressed gene can complete RNA chain every 10
sec.)
2.4.3 Termination
The termination of transcription is different for the three different eukaryotic RNA polymerases.
Transcription of pre-rRNA genes by RNA polymerase I is terminated by a process that makes use
of a polymerase-specific termination factor. This factor is a DNA-binding protein that binds
downstream of the transcription unit (unlike the E. coli Rho factor, which is a RNA-binding
termination factor).
Purified RNA polymerase III terminates transcription after polymerizing a series of U residues;
Termination by RNA polymerase III however does not require an upstream stem-loop secondary
structure in the RNA transcript (as in bacteria).
Termination of transcription by RNA pol II occurs at a point after the end of the protein coding
sequence by a mechanism which is not very clear. Termination signals are difficult to identify
because the 3´ end of the transcript is removed shortly after it is synthesized. It is also possible
that the dissociation of a transcription factor destabilizes the transcription complex causing the
RNA polymerase to dissociate from the template later.
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2.5 Post-transcriptional Modifications of mRNA
In eukaryotic cells, the primary RNA transcripts are processed and converted into mature RNA
by post transcriptional modifications. Post transcriptional modification takes place prior to
translation. Pre-mRNA molecules are processed to mature mRNA by 5´capping, 3´clevage and
polyadenylation, splicing and methylation.
The coding information in eukaryotic genes is discontinuous and is arranged as a series of exons
separated by non-coding sequences or introns (transcribed, not translated). RNA splicing is the
process by which introns or the intervening sequences present in the primary transcript (premRNA) are removed and the exons (coding regions) are then joined to form a single continuous
RNA molecule. In most cases, RNA splicing occurs after the completion of transcription and
end-capping of the pre-mRNA; however transcripts can be spliced co-transcriptionally. The
process ensures that the mRNA is an accurate template for protein synthesis. Further, the 5´end
of the RNA is altered by the addition of a modified nucleotide, 7 methyl guanosine by a process
called capping and the 3´end is modified by the addition of a poly A tail of upto 250 adenines, a
process called polyadenylation.
RNA transcribed by RNA pol II exists in the nucleus as a population of molecules of different
lengths and at different stages of processing. These are known collectively as heterogenous
nuclear RNA (hnRNA).
2.5.1 Splicing- This process takes place in the nucleus and involves the removal of non-coding
intron sequences from pre-mRNA and joining of coding sequences exons to produce mature
mRNAs. The mature mRNA consists of the coding sequences corresponding to the exons which
are continuous. The mRNA is then exported to the cytoplasm where it acts as a template for
protein synthesis.
Splicing depends on the presence of signal sequences in the pre-mRNA. In most of the genes, the
first two nucleotides at the 5´ end of an intron are GU and the last two at the 3´ end are AG (Fig.
9). These are part of larger signal sequences present at the 5´ and 3´ ends of the introns. The
complete 5´ signal sequence is 5´AGGTAAGT3´ and the 3´sequence is 5´YYYYYYNCAG3´(Ypyrimidine; N-any nucleotide). In addition to these, the vertebrates have a sequence, 5´
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CURAY3´ (R-purine, Y pyrimidine) which is called the branchpoint sequence. This sequence is
present in the intron 10-40 bases upstream of the 3´ signal sequence. Splicing occurs in two
steps. In the first step the 2´ hydroxyl group of adenine of the branchpoint sequence attacks the
phosphodiester bond 5´ to the G of GU (5´ splice site). The bond is broken releasing the 5´ end
of the intron. This 5’ end forms a 2’ 5’ phosphodiester linkage with the branchpoint sequence.
This forms a tailed loop structure called a “LARIAT”. In the second step, the 3´ end of the intron
is cleaved after the G of the AG (3´splice site).The intron is now released and the two exons are
joined together.
Fig 9. Splicing of eukaryotic pre-mRNA-Molecular mechanism
Splicing is catalyzed by a group of molecules called “small nuclear ribonucleoproteins” (snRNPs
or snurps). These are composed of small RNA molecules rich in uracil called URNA or small
nuclear RNAs (snRNAs) that are complexed with proteins. Many types of snRNPs exist but the
most abundant are U1, U2, U4, U5 and U6. Each snRNP contain a single URNA molecule,
except U4 and U6 (which exist base paired to each other in the same snRNP). The RNA
component of the snRNPs interacts by base pairing with splicing signal sequences of pre mRNA.
The U1 snRNP binds to the 5´ splice site and the U2 snRNP binds to the branchpoint sequence of
intron. The remaining snRNPs, U5 and U4/U6 then form a complex with U1 and U2 causing the
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intron to loop out and the two adjacent exons to be brought together. The combination of premRNA and snRNPs is called the spliceosome. This is responsible for folding of pre-mRNA in
the correct conformation for splicing. The spliceosome also catalyzes the cutting and joining
reactions that excise the intron and ligate the exons. Once splicing is completed, the spliceosome
dissociates (Fig 10). The functioning of the spliceosome is not fully understood and the
components responsible for all its enzymatic activities have not been identified.
Fig. 10 Splicing of eukaryotic pre-mRNA-role of snRNP
2.5.2 Capping
Eukaryotic mRNAs are altered at their 5´ end by a modification known as capping which
involves addition of the modified nucleotide, 7-methyl guanosine (m7G) (Fig 11). The cap is
added by the enzyme guanyltransferase which joins GTP by an unusual 5´-5´ triphosphate
linkage to the first nucleotide of the mRNA (in reverse orientation). Methyl transferase then adds
a –CH3 (methyl) group to the nitrogen at 7 position of guanine ring. The enzyme usually adds
methyl groups to 2´ hydroxyl group on the ribose sugar of next two nucleotides in the mRNA.
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Transcription
The cap structure forms a barrier to 5´ exonuclease activity. It thus stabilizes the transcript in the
cytoplasm. This also provides to the ribosome to recognize the start of a mRNA molecule.
Fig 11. Capping of the 5′ end of nascent RNA transcripts
with 7′-methylguanylate (m7G)
(Source:http://en.wikipedia.org/wiki/Five-prime_cap)
2.5.3 3´Cleavage and Polyadenylation
The process of polyadenylation begins on the completion of the process of transcription. The 3'
end sequences in most segment of the newly made pre-mRNA is first cleaved off by a set of
proteins. Following cleavage, these proteins then synthesize the poly (A) tail at the 3' end of
RNA.
In some genes, poly (A) tail may be added at any one of several possible sites. Hence,
polyadenylation can produce more than one transcript from a single gene (alternative
polyadenylation).
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Transcription
The cleavage and polyadenylation reaction requires that a specific sequence be present in the
pre-mRNA transcript. This consists of 5´AAUAAA3´, the polyadenylation signal, followed by a
5´YA3´ where Y-pyrimidine in next 11-20 nt. Downstream a GU-rich sequence is often present
(Fig. 12). Collectively these sequences are important for polyadenylation. A number of specific
protein factors recognize these sequences and bind to the pre-mRNA and form a complex. When
the complex has assembled, cleavage takes place and then one of the factors, poly (A)
polymerase (PAP), adds upto 250 A residues to the 3´ end of the cleaved pre-mRNA. The poly
(A) tail is very important for the nuclear export of the transcript, translation, and stability of
mRNA. It has been seen that the tail gets shortened over time, and, when it is significantly
shortened, the mRNA is enzymatically degraded. However some mRNAs, notably those
encoding histone proteins, have no poly (A) tail.
Fig. 12 Cleavage and Polyadenylation of Eukaryotic pre-mRNAs
CPSF - Cleavage and Polyadenylation Specificity Factor; CStF - Cleavage Stimulatory Factor
2.5.4 Pre-mRNA Methylation
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Transcription
A small percentage of A residues in pre-mRNA, those in the sequence 5´-RRACX-3´(R-purine
and X is rarely G) becomes methylated at 6 position. Methylation seems to be largely conserved
in the mature mRNA, though their function is unknown.
2.6 Stability of m-RNA
The mRNA is relatively unstable as compared with rRNA or tRNA. Therefore cells regulate
protein levels by altering the rate of gene transcription. Prokaryotic mRNA has a much shorter
half-life then eukaryotic mRNA. In E.coli, generally the half-life of mRNA is about 2 minutes.
While in many other bacterial strains the half-life of mRNA may range from 40 seconds to about
60 seconds, in case of Eukaryotes, the half-life of mRNAs can be of several days.
3. Inhibitors of transcription
Transcription inhibitors have great importance as they can be used as antibiotics against,
pathogenic bacteria. An example of such an antibacterial or anti-tuberculosis drug is rifampicin,
which inhibits transcription in prokaryotes by inhibiting DNA-dependent RNA polymerase.
Rifampicin binds to beta-subunit of RNA polymerase. A very important inhibitor of transcription
is an antifungal, 8-Hydroxyquinoline. The chemicals which alter histone methylation may also
act as transcription inhibitors. α-Amanitin and actinomycin D are also used to inhibit eukaryotic
DNA transcription. α-Amanitin gets bound to RNA polymerase II and III while actinomycin D
intercalates between the DNA bases and hence hinder the progress of RNA polymerase.
Summary of the Chapter
Transcription is the first step of gene expression that involves synthesis of RNA from a DNA
template by the enzyme RNA polymerase. In the bacterial system, a single RNA polymerase
(RNAP) is responsible for synthesis of all types of RNAs, while in the eukaryotic system there
are five different types of RNA polymerases that are involved in transcription, although type IV
and V polymerase are exclusively present in plants. Transcription is divided into various phases
i.e. pre-initiation, initiation, promoter clearance, elongation and termination. Unlike prokaryotes,
in eukaryotes, post transcriptional modifications convert primary transcripts into mature RNA.
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Transcription
Quiz-1: Multiple Choice Questions
1.
A DNA strand (template strand) has the sequence 3’TTACGACGG5’, what would be
the sequence of the RNA strand transcribed from the DNA?
a) 5’AAUGCUGCC3’
b) 5’UGGCGACCC3’
c) 5’GCAUCAACC3’
d) 5’AAGCGCACC3’
2.
Complete the statement-Once a strand of mRNA starts being translated
a) DNA transcription shuts down
b) another mRNA is made simultaneously from the DNA
c) only a single protein will be transcribed
d) it is degraded so that only one polypeptide is made from this mRNA
3.
Which one of the following statements about E. coli RNA polymerase is false?
a)
b)
c)
d)
4.
The specificity of RNA polymerase for specific genes is governed by
a)
b)
c)
d)
5.
Alpha- factor
Sigma factor
Beta factor
Omega factor
Which of the following statements is not correct?
a)
b)
c)
d)
6.
The holoenzyme includes the sigma factor
The core enzyme includes the sigma factor
It requires Mg2+ for its activity
It requires Zn2+ for its activity
There are two alpha subunits in the E.coli RNA polymerase
There is one beta subunit in the E.coli RNA polymerase
RNA polymerase of E.coli has only one type of sigma factor
The beta subunit of E. coli RNA polymerase is inhibited by rifampicin
Which of the following best explains the sequence of gene expression?
a) DNA to RNA to protein
b) RNA toDNA to protein
c) DNA to protein to RNA
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Transcription
d) Protein to DNA to RNA
7.
RNA polymerase II transcribes all of the following genes except genes specifying
_____.
a)
b)
c)
d)
8.
rRNAs
proteins
miRNAs
snRNAs
What is a promoter?
a) An extracellular environmental inducer that controls genes expression
b) A regulatory protein that accelerates mRNA turnover
c) An element that promotes termination of transcription
d) A specific target sequence to which RNA polymerase binds
9.
Some eukaryotic promoters contain an element positioned around nucleotide +1 called
a)
b)
c)
d)
the Pribnow box
an initiator (Inr) sequence
an inverted repeat
the TATA box
Quiz-2: Match the Following1
α-amanitin
a
TATA binding protein
2
Pribnow box
b
Tailed loop structure
TBP
c
d
5
6.
polymerase-DNAnascent RNA
LARIAT
RNA polymerase II
Ternary Elongation
Complex
TATAAT
7
8
9
TFIIH
C-terminal domain
TFIIB
g
h
i
3
4
e
f
Amatoxins
Attaches to TATA
binding protein
5’-TTGACA-3’
-25 TATA box
Phosphorylation site in
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Transcription
10 -35 box
j
RNA polymerase II
Helicase activity
Key to Quiz-2
1-e
2-d
3-a
4-c
5-b
6-h
7-j
8-i
9-f
10-g
Quiz3- Long answer questions- (refer to the chapter for complete answers)
1. Describe the structure of E.coli RNA polymerase and discuss the roles of different
components of the enzyme in transcription.
2. Describe the three eukaryotic RNA polymerases and their role in transcription.
3. What are Transcription Factors? How are they involved in the process of eukaryotic
transcription?
4. Describe the process of formation of preinitiation transcription complex in
eukaryotes.
5. Write short notes on a) rho factor b) Pribnow box c) TATA box
Glossary
1. α-amanitin-is a cyclic peptide of eight amino acids, most deadly of all the amatoxins
(toxins found in several species of the Amanita genus of mushrooms). α-Amanitin is an
inhibitor of RNA polymerase II and III. Hence it can also be used to determine which
types of RNA polymerase are present.
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2. Intron-Intervening nucleotide sequence within a gene that is removed during the
process of RNA splicing when the final mature RNA product of a gene is being
generated.
3. Promoter- DNA region to which RNA polymerase binds when initiating transcription.
4. RNA Polymerase-(RNAP or RNA pol), also known as DNA-dependent RNA
polymerase, is an enzyme that produces primary transcript RNA
5. Splicing-Precise ligation of exon by excision of the intervening sequences from an RNA
primary transcript. A functional m RNA molecule is formed after splicing. RNA splicing
is a post transcriptional process in eukaryotes
6. Transcription- Transfer of information from DNA to RNA.
Further Readings:
1. Pribnow, David (1975) Nucleotide sequence of an RNA polymerase binding site at an
early T7 promoter PNAS 72 (3): 784–788
2. Alberts B, Bray D, Lewis J, RaffM, Roberts K, and Watson JD (1994) Molecular Biology
of the Cell, (3rded.) Garland Publishing, New York.
3. Burley SK (1996) The TATA box-binding protein. Curr.Opin.Struct. Biol. 6, 69-75
4. Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, and Darnell J (2000)
Molecular Cell Biology (4thed.) W. H. Freeman, New York
5. Alberts B, Bray D, Hopkin K, Johnson A, Lewis J, Raff M, Roberts K, Walter
P(2004)Essential Cell Biology(2nd ed.) Garland Science ,New York
6. Turner PC, McLennan AG, Bates AD, and White MRH (2001). BIOS Instant notes in
Molecular Biology (2nded.)Bios Scientific Publishers Ltd. Oxford, UK
7. Wikipedia, the free encyclopedia-www.wikipedia.org
http://en.wikipedia.org/wiki/Bacterial_transcription;
http://en.wikipedia.org/wiki/Eukaryotic_transcription;
http://en.wikipedia.org/wiki/Transcription_factor
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