1

Transcription in Prokaryotes and

Eukaryotes

[Manuscript]

Subject:- Zoology

Course:- BG 4th Semester

Paper:- Core Course 4, Genetics and

Evolutionary Biology

Topic No:- 2.3 –Transcription

Lecture:- Transcription in Prokaryotes

and Eukaryotes
 2

Introduction
The genetic code in DNA is frequently referred to as a "blueprint"
because it contains the instructions a cell requires in order to sustain itself
or we can say “DNA is a book of life”. The instructions stored within DNA
are "read" in two steps: transcription and translation. In transcription, a
portion of the double-stranded DNA template gives rise to a single-
stranded RNA molecule. In some cases, the RNA molecule itself is a
"finished product" that serves some important function within the cell.
Often, however, transcription of an RNA molecule is followed by a
translation step, which ultimately results in the production of a protein
molecule. These instructions flow from DNA into RNA and RNA into
protein.
The first step in this gene expression is transcription where genetic
information in a strand of DNA is copied and reconstructed in the form of
a new molecule of RNA. This copying of information from DNA and
rewriting it in the form of RNA is called Transcription. Actually
transcription is the process of synthesis of RNA from DNA. All RNA
molecules except the RNA genomes of certain viruses are derived from
information permanently stored in DNA. During transcription, an enzyme
system converts the genetic information in a segment of double-stranded
DNA into a single stranded RNA.
Through transcription three major kinds of RNA are produced. Messenger
RNA (mRNA), Transfer RNA (tRNA) and Ribosomal RNA (rRNA).
Messenger RNA (mRNA) contains the information of amino acids
sequence in proteins to be synthesized. Transfer RNA (tRNA) reads the
information encoded in the mRNA and transfers the appropriate amino
acid to a growing polypeptide chain during protein synthesis. Ribosomal
RNAs (rRNA) are constituents of ribosomes, the cellular machines that
synthesize proteins. Many additional, specialized RNAs have regulatory
or catalytic functions or are precursors to the three main classes of RNA.
The sum of all the RNA molecules produced in a cell under a given set of
conditions is called the cellular transcriptome.
Much of the genome of humans and other mammals is transcribed into
RNA. The products are predominantly not mRNAs, tRNAs, or rRNAs, but
rather special-function RNAs, a host of which are being discovered.
The process of transcription begins when an enzyme called RNA
polymerase (RNA pol) attaches to the template DNA strand and begins
to catalyze production of complementary RNA. Polymerases are large
enzymes composed of multiple subunits, and when active on DNA, they
are also typically complexed with other factors.
 3

Description
Transcription is DNA dependent RNA synthesis. RNA is synthesized on
DNA template. RNA is single stranded but DNA is double stranded. The
information from only one strand of DNA is copied into RNA. The DNA
strand on which RNA is synthesized is called template strand and the
other strand of DNA is called non-template strand with respect to
transcription process. The RNA formed is identical in sequence with the
non-template strand. The non-template strand is also called coding
strand, because the RNA formed is identical in sequence with the non-
template strand. The DNA strand is copied in 3'-to-5' direction
antiparallel to RNA strand which builds in 5'-to-3' direction.

Fig 1.1- Template and Non-template strands of DNA

The transcribed segment of DNA is called a transcription unit. This unit

is a sequence of nucleotides in DNA that codes for a single RNA molecule,
containing a promoter, an RNA-coding sequence, and a terminator. In
eukaryotes, a transcription unit typically carries the information of just one
gene and it is termed as monocistronic transcription unit. In prokaryotes,
a set of adjacent genes is often transcribed as a unit termed polycistronic
transcription unit. The immediate product of transcription is called the
primary transcript. First base pair from which transcription starts is called
the start point or transcription initiation site. From this point, RNA
polymerase moves along the template, synthesizing RNA, until it reaches
a terminator sequence. Sequences prior to the start point are described
 4

as upstream of it designated by + sign, those after the startpoint (within

the transcribed sequence) are downstream of it designated by - sign

Fig 1.2- Transcription unit

An enzyme system converts the genetic information from the transcription

unit of double stranded DNA into a single stranded RNA. The main
enzyme involved in transcription is RNA polymerase (RNA pol). This
enzyme joins the transcription unit at promoter region, then moves along
the RNA coding region to copy the information from template strand to
form primary transcript and then leaves the unit at terminator region. RNA
polymerases have been found in all species, but the number and
composition of these proteins vary across taxa. For instance, bacteria
contain a single type of RNA pol while eukaryotes (multicellular organisms
and yeasts) contain three distinct types. Each RNA polymerase is a
complex molecule consisting of many protein subunits.
In E .Coli bacteria (Prokaryote) RNA pol is a multi-subunit enzyme made
up of five different polypeptides – α, β, β’, ω, σ.

Fig 1.3 – RNA polymerase in E. Coli

 5

The holoenzyme (α2ββ’ω σ) can be separated into two components, the

core enzyme (α2ββ’ω) and the sigma factor (the σ polypeptide).
The transcriptional machinery in the nucleus of a eukaryotic cell is much
more complex than that in bacteria. Eukaryotes have three RNA
polymerases, designated I, II, and III, which are distinct complexes but
have certain subunits in common. Each polymerase has a specific
function. RNA polymerase I (Pol I) is responsible for the synthesis of only
one type of RNA, a transcript called pre-ribosomal RNA (or pre-rRNA),
which contains the precursor for the 18S, 5.8S, and 28S rRNAs. The
principal function of RNA polymerase II (Pol II) is synthesis of mRNAs
and some specialized RNAs. RNA polymerase III (Pol III) makes tRNAs,
the 5S rRNA, and some other small specialized RNAs.
DNA-dependent RNA synthesis, in addition to a DNA template and RNA
polymerase, requires all four ribonucleoside triphosphates as precursors
of the nucleotide units of RNA, as well as Mg2+ ions. These four precursors
are adenosine triphosphate (ATP), guanosine triphosphate (GTP),
cytidine triphosphate (CTP) and uridine triphosphate (UTP).
The transcription process starts from the promoter region of transcription
unit. First of all RNA pol binds the promoter region. Then RNA pol
polymerizes first few ribonucleotides on the template strand to initiate the
process. After initiation the RNA is elongated through elongation process
where RNA pol moves downstream along the template strand and adds
new ribonucleotide units to the growing RNA strand. When RNA pol
reaches the termination site, both RNA pol and RNA transcript leaves the
transcription unit resulting into termination of transcription process.

Fig 1.4 – General overview of transcription process.

 6

Transcription in prokaryotes
In prokaryotes transcription process is completed in 3 phases: Initiation,
Elongation and Termination. All these phases are described in detail
separately.
1. Initiation.
The first step in transcription is initiation. In this phase the RNA pol binds
to the transcription unit to be transcribed at a specialized sequence called
a promoter.
In prokaryotes (E. coli), promoters are usually composed of three
sequence elements. Most genes have a sequence called the Pribnow
box, with the consensus sequence TATAAT positioned about 10 base
pairs upstream from the site that serves as the location of transcription
initiation. Many genes also have the consensus sequence TTGCCA at a
position 35 bases upstream of the start site, and some have what is called
an upstream (UP) element, which is an A-T rich region 40 to 60
nucleotides upstream that enhances the rate of transcription.

Fig 1.5 – Promoter region in E. coli.

In any case, upon binding, the RNA pol "core enzyme" (α2ββ’ω) binds to
another subunit called the sigma (σ) subunit to form a holoenzyme
(α2ββ’ω σ) capable of unwinding the DNA double helix in order to
facilitate access to the gene. It is the sigma (σ) subunit which conveys
promoter specificity to RNA polymerase; that is, it is responsible for telling
RNA polymerase where to bind. There are a number of different sigma
subunits that bind to different promoters and therefore assist in turning
genes on and off as conditions change.
The pathway of transcription initiation consists of two major parts, binding
and initiation, each with multiple steps. First, the polymerase, directed by
its bound σ factor, binds to the promoter. In the binding phase, the initial
interaction of the RNA polymerase with the promoter leads to formation of
a closed complex, in which the promoter DNA is stably bound but not
unwound. A 12 to 15 bp region of DNA—from within the -10 region to
position +2 or +3 is then unwound to form an open complex and a
transcription bubble is created.
 7

Fig 1.6 – Steps involved in initiation process in E. coli.

Unlike DNA polymerase, RNA polymerase does not require a primer to

initiate synthesis. Initiation occurs when RNA polymerase binds at
promoters. RNA polymerase then starts to synthesize the initial DNA-RNA
heteroduplex, with ribonucleotides base-paired to the template DNA
strand according to Watson-Crick base-pairing interactions. RNA
polymerase forms an RNA strand by adding ribonucleotides, building RNA
in the 5'-to-3' direction. Beta (β, β’) subunits form the catalytic centre of
RNA pol enzyme.
 8

2. Elongation.
RNA polymerase interaction with the promoter region inhibits the
enzyme's ability to access DNA further downstream and thus the
synthesis of the full-length product. In order to continue RNA synthesis,
RNA polymerase must escape the promoter. When transcription is
initiated within the complex, it leads to a conformational change that
converts the complex to the elongation form, followed by movement of the
transcription complex away from the promoter (promoter clearance). The
σ subunit dissociates at random as the polymerase enters the elongation
phase of transcription. The protein NusA binds to the elongating RNA
polymerase to carry on the elongation process.

Fig 1.7 – Steps involved in elongation process in E. coli.

During the elongation phase of transcription, the growing end of the new
RNA strand base-pairs temporarily with the DNA template to form a short
hybrid RNA-DNA double helix, estimated to be 8 bp long. The RNA in
this hybrid duplex “peels off” shortly after its formation, and the DNA
duplex re-forms. During transcription, the E. coli RNA polymerase
generally keeps about 17 bp unwound in the transcription bubble. The 8
bp RNA-DNA hybrid occurs in this unwound region. Elongation of a
transcript by E. coli RNA polymerase proceeds at a rate of 50 to 90
nucleotides/s. Because DNA is a helix, movement of a transcription
bubble requires considerable strand rotation of the nucleic acid molecules.
 9

As a result, a moving RNA polymerase generates waves of positive

supercoils ahead of the transcription bubble and negative supercoils
behind. These supercoils are relieved through the action of enzyme
topoisomerase.

Fig 1.8 –Elongation process in E. coli.

3. Termination.
When transcription is complete, the primary transcript of RNA is released,
the NusA protein dissociates, and the RNA polymerase dissociates from
the DNA. Another σ subunit binds to the RNA polymerase and the process
begins again.

Fig 1.9 –Termination of transcription in E. coli.

 10

An encounter with certain DNA sequences results in a pause in RNA

synthesis, and at some of these sequences transcription is terminated. In
bacteria. E. coli there are at least two classes of termination signals
(Sequences): one class relies on a protein factor called ρ (rho) and the
other is ρ independent.
Most ρ -independent terminators have two distinguishing features. The
first is a region that produces an RNA transcript with self-complementary
sequences, permitting the formation of a hairpin structure. The second
feature is a highly conserved string of three A residues in the template
strand that are transcribed into U residues near the 3' end of the hairpin.
When a polymerase arrives at a termination site with hairpin structure, it
pauses. Formation of the hairpin structure in the RNA disrupts several A-
U base pairs in the RNA-DNA hybrid segment and may disrupt important
interactions between RNA and the RNA polymerase, facilitating
dissociation of the transcript and hence termination of the whole process.

Fig 1.10 –Mechanism of Termination in E. coli.

 11

The ρ -dependent terminators lack the sequence of repeated A residues

in the template strand but usually include a CA-rich sequence called a rut
(rho utilization) element. The ρ protein associates with the RNA at rut
sites and migrates in the 5'-to-3' direction until it reaches the transcription
complex that is paused at a termination site. Here it contributes to release
of the RNA transcript.

Transcription in Eukaryotes
In both prokaryotes and eukaryotes transcription process is completed in
3 phases: Initiation, Elongation and Termination.
However the transcriptional machinery in the nucleus of a eukaryotic cell
is much more complex than that in bacteria. Eukaryotes have three RNA
polymerases, designated I, II, and III, which are distinct complexes but
have certain subunits in common. Each polymerase has a specific
function and is recruited to a specific promoter sequence. Pol I promoters
differ greatly in sequence from one species to another. Some Pol II
promoters have a few sequence features in common, including a TATA
box near base pair -30 and an Inr sequence (initiator) near the RNA
start site at +1.

Fig 1.11 –Promoter region for RNA pol II in Eukaryotes.

RNA polymerase II is central to eukaryotic gene. This polymerase is

strikingly more complex than its bacterial counterpart. Pol II from yeast is
a huge enzyme with 12 subunits. The largest subunit (RBP1) exhibits a
high degree of homology to the β’ subunit of bacterial RNA polymerase.
Another subunit (RBP2) is structurally similar to the bacterial β subunit,
and two others (RBP3 and RBP11) show some structural homology to
the two bacterial α subunits. Pol II must function with genomes that are
more complex and with DNA molecules more elaborately packaged than
in bacteria. RNA polymerase II requires an array of other proteins, called
transcription factors, in order to form the active transcription complex. The
general transcription factors required at every Pol II promoter (usually
designated TFII) are highly conserved in all eukaryotes. The largest
subunit of Pol II (RBP1) also has an unusual feature, a long carboxyl-
 12

terminal tail called carboxyl-terminal domain (CTD). The CTD has many
important roles in Pol II function, as outlined below.
The process of transcription by Pol II can be described in terms of several
phases—assembly, initiation, elongation, termination—each
associated with characteristic proteins.
Assembly of RNA Polymerase and Transcription Factors at a
Promoter
The formation of a closed complex begins when the TATA-binding
protein (TBP) binds to the TATA box. TBP is bound in turn by the
transcription factor TFIIB, which also binds to DNA on either side of TBP.
TFIIA binds, and along with TFIIB helps to stabilize the TBPDNA
complex. TFIIB provides an important link to DNA polymerase II, and the
TFIIB-TBP complex is next bound by another complex consisting of TFIIF
and Pol II. TFIIF helps target Pol II to its promoters, both by interacting
with TFIIB and by reducing the binding of the polymerase to nonspecific
sites on the DNA. Finally, TFIIE and TFIIH bind to create the closed
complex. TFIIH has multiple subunits and includes a DNA helicase activity
that promotes the unwinding of DNA near the RNA start site, thereby
creating an open complex.
RNA Strand Initiation and Promoter Clearance
TFIIH has an additional function during the initiation phase. A kinase
activity in one of its subunits phosphorylates Pol II at many places
includind CTD. This causes a conformational change in the overall
complex, initiating transcription. During synthesis of the initial 60 to 70
nucleotides of RNA, first TFIIE and then TFIIH is released, and Pol II
enters the elongation phase of transcription.
Elongation, Termination, and Release
TFIIF remains associated with Pol II throughout elongation. During this
stage, the activity of the polymerase is greatly enhanced by proteins called
elongation factors. The elongation factors, suppress pausing during
transcription and also coordinate interactions between protein complexes
involved in the post transcriptional processing of mRNAs. Once the RNA
transcript is completed, transcription is terminated. Pol II is
dephosphorylated and recycled, ready to initiate another transcript.
The primary transcript of RNA produced through this process needs
further processing in order to function properly.
 13

Fig 1.12 –Different phases of transcription in eukaryotes

 14

Transcription in prokaryotes and eukaryotes is compared in the following

table.
Prokaryotes Eukaryotes
 Transcription occurs in  Transcription occurs in
cytoplasm of prokaryotic cell. nucleus of eukaryotic cell.
 Single RNA pol have  3 classes of RNA
capability to produce r RNA, polymerases are involved.
mRNA and tRNA. Pol I for rRNA, Pol II for
mRNA and Pol III for tRNA.
 RNA produced is fully  RNA produced needs further
functional for translation processing to become fully
process. functional.
 Transcription along with  After transcription is
translation process take complete then only
place simultaneously. translation takes place.
 Transcribed RNA is  Transcribed RNA is
polyscistronic, i.e multiple monocistronic, i.e single
genes are regulated by gene is regulated by single
single promoter. promoter.
 Termination needs Rho  Termination does not need
factor. Rho factor.
 Needs sigma factor for RNA  Needs a set of proteins
pol to bind to promoter. called transcriptional factors
for RNA pol to bind to
promoter.