Chapter 4

Mechanism of Transcription in
Prokaryotes

Abstract
Transcription is a process of reading and expressing the information of DNA
(Deoxyribose nucleic acid) in the form of RNA (Ribose nucleic acid/ Ribonucleic
acid). RNA is also a polynucleotide chain like DNA but with little discriminations.
RNAs commonly exist as single stranded whereas DNA is double stranded. Contrary
to DNA, in RNA, deoxyribose is replaced by ribose and in place of thymine, uracil
is present. However, different types of RNA have been found in a cell such as,
messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA),
microRNA, SnRNA, SnoRNA, Xist and Tsix etc. Among these, the major types of
RNAs are; mRNA, rRNA, and tRNA. The mechanism of prokaryotic transcription is
simple. In prokaryotes, such as E. coli, only one type of DNA dependent RNA
polymerase is present. This species of polymerase is specific for the transcriptional
process in E. coli. All RNA polymerases, whether prokaryotic or eukaryotic, do not
require primers to start transcription process. In prokaryotes, there is no
compartmentalization; transcription and translation take place in same compartment
that is basically a single cell organism.
Keywords: Transcription, prokaryotes, degradosome, RNA polymerase, promoter
strength

4.1 Introduction
Transcription is a process of RNA synthesis from DNA. In prokaryotes, transcription
and translation processes take place in the same chamber. Only one kind of RNA
polymerase is involved for transcription in prokaryotes and it consists of five
subunits of four types (α2ββ́σ). Sigma (σ) subunit of RNA polymerase recognizes
promoter regions on DNA strand. There are mainly two promoter regions in case of
prokaryotes that are present at -10bps (pribnow box) and -35bps (recognition box)
upstream to transcriptional start site. In many cases, before the start of the gene
sequence (coding for protein) a leader sequence (5́ UTRs/ untranslated regions) and
at the end of a gene, a trailer sequence (3΄UTRs/untranslated regions) are transcribed
by RNA polymerase. Two types of termination signals are present in E. coli, one is
ρ dependent termination signal and the other is ρ independent termination or intrinsic
termination signal. However, the population of mRNA in a cell is mainly dependent

37
38 Chapter 4

on the rate of biogenesis of mRNA and then its degradation rate. Thus degradosome
decays RNA and acts as RNA decay machine that also maintains RNA turnover rate.

4.2 RNA polymerase of prokaryotes

RNA polymerase of E. coli is a multimeric enzyme consisting of five subunits of
four kinds i-e; α2ββ́σ (Fig 4.1). These subunits of RNA polymerase are encoded by
rpoA, rpoB, rpoC and rpoD genes respectively. RNA polymerase with its complete
subunits set is called holoenzyme (α2ββ́σ) with its specific recognition and catalytic
activity, whereas without σ subunit this enzyme is called core enzyme (α2ββ́)
possessing only catalytic roles. The catalytic site of RNA polymerase runs a catalytic
mechanism in which two active metal ions (Mg2+ ions) are involved. These metal
ions interact and bind with three Asparate (Asp) residues of RNA polymerase, which
are highly conserved residues (Murakami and Darst 2003; Nelson and Cox 2012).
By binding with Asp residues, one Mg2+ ion mediates the nucleophilic attack of 3́ –
OH group to α phosphate of incoming nucleotide and second Mg2+ ion interacts with
pyrophosphates and facilitates its removal. This Mg2+ ion is also released along with
pyrophosphates and a thermodynamically favorable reaction takes place. This
process of catalytic mechanism is common in all types of polymerases.

Fig. 4.1 Subunits of RNA polymerase of E. coli (Modified from Geszvain and
Landick 2005)

The largest subunit of RNA polymerase is β́, which plays a role in DNA binding
while σ subunit recognizes and binds specific sequences or signals on DNA called
promoters. Two αsubunits are present in RNA polymerase those recognize UP
elements (upstream elements in case of rRNA gene) along with activation and
assembling of enzymes as well as other regulatory factors (Geszvain and Landick
2005; Nelson and Cox 2012).

4.3 Transcription process

Like DNA replication, transcription process also involves three main steps;
Initiation, Elongation and Termination.
Mechanism of Transcription in Prokaryotes 39

4.3.1 Transcription initiation

Transcription initiation starts with the recognition of specific signals on DNA strand
(present in non-template strand) by σ subunit of RNA polymerase. In transcription,
both DNA strands do not serve as template. One strand that runs towards 3´----
>5´direction serves as template and is known as antisense or non-coding strand,
whereas its counterpart that runs in 5´---->3´direction is called non-template/sense or
coding strand because RNA transcript is identical to this DNA strand.
4.3.1.1 Promoter regions of genes
In prokaryotes, there are commonly two promoters present at -10 bps (5´ TATAAT
3´) and -35 bps (5´ TTGACA 3´) upstream to the transcriptional start site. The former
promoter sequence is called -10 box or pribnow box whereas the later is designated
as -35box or recognition box (Fig 4.2) (Watson et al. 2004; Lodish et al. 2012).

Fig. 4.2 Promoter regions of genes in prokaryotes (Conceived from Watson et al.
2004)

Pribnow box and recognition box are separated from each other by ~17 bps non-
conserved sequences (Fig 4.3).

Fig. 4.3 Non-conserved sequence between -10 box and -35 box (promoter regions)
(Modified from Watson et al. 2004)

4.3.1.2 Promoter strength

The promoter strength is dependent on the length of non-conserved sequence that
separates -10 box and -35 box promoter regions; hence the distance between these
two boxes affects the promoter strength. The promoter strength is also affected by
40 Chapter 4

the closeness and similarity of -10 box and -35 box promoter regions to their
consensus sequences. If pribnow box and recognition box sequences of promoter are
close to their respective consensus sequences then these promoters are said to be
strong promoters and would have more strength than those, whose -10 box and -35
box sequences have more substitution and are referred as weak promoters (Watson
et al. 2004; Lodish et al. 2012).

4.3.2 Transcription Elongation

When transcription starts in prokaryotes e.g. E.coli,RNA polymerase (holoenzyme)
first recognizes and binds to -35box and drape ~60bps including -10 bps and -35 bps
sequences forming a closed promoter complex (Fig 4.4). DNA starts to melt and
unwind at pribnow box that is rich in A═T sequence and thus DNA duplex
dissociates, open promoter complex is established and σ subunit leaves the RNA
polymerase. This core enzyme (RNA polymerase without σ subunit) then
translocates on DNA strand and dictates the information of DNA into RNA
transcript. The addition of first two nucleotides results in the formation of first
phosphodiester bond and an elongation step starts to proceed.

Fig 4.4 RNA polymerase attachment to promoter regions for transcription.

(Redrawn after modication from Brown 1998)

In many cases, before the start of the gene sequence (coding for protein), a leader
sequence is transcribed. Similarly, at the end of a gene, a trailer sequence is also
transcribed by RNA polymerases. These leader and trailer sequences are of variable
lengths that may vary in every case. These are also called untranslated regions (UTR)
and are 5́ UTRs (leader sequences) and 3΄UTRs (trailer sequences) (Brown 1998;
Nelson and Cox 2012).
4.3.2.1 Polymerization of ribonucleotides in Transcription Elongation
RNA polymerases add ribonucleotides, complementary to DNA template into RNA
polymers. Polymerization of ribonucleotides occurs in the same way as the addition
of deoxyribonucleotides takes placein DNA replication. Ribonucleotides are added
or joined, when 3΄-OH end of first ribonucleotide does a nucleophilic attack on α
phosphate of coming ribonucleotide. Resultant to this, pyrophosphates are released
and phosphodiester bond between these ribonucleotides is established (Watson et al.
2004; Nelson and Cox 2012).
Mechanism of Transcription in Prokaryotes 41

4.3.3 Transcription Termination

The synthesis of RNA continues until a transcription termination signal is reached.
Two types of transcription termination signals are present in E. coli. One of these
signals relies on a protein known as ρ (rho) and hence termination involving this
signal is known as ρ dependent termination while the other signal does not rely on ρ
(rho), and the process is called ρ independent termination or intrinsic termination. In
transcription termination of E.coli, the formation of stem loop structure due to
pallindromic sequences is a common attribute. Pallindromic sequences are G ≡ C
rich inverted sequences that make the stem loop structure stable. These inverted
repeats of pallindromic sequence are separated by a non-repeated sequence (Brown
1998; Watson et al. 2004).
4.3.3.1 ρ independent termination
The facet of ρ independent termination is that the pallindromic sequences of template
strand are followed by repeat of ~10As residues. As the pallindromic sequence is
transcribed and stem loop structure is formed, the RNA polymerase halts and stops
its translocation. At the same time, RNA polymerase does the addition of Us that are
complementary to run off As in the template. The dissociation of transcript takes
place with the breakage of A=U base pairing in DNA:RNA duplex, so RNA
transcript dissociates from the template (Lewin 2004; Lodish et al. 2012; Nelson and
Cox 2012)
4.3.3.2 ρ dependent termination
In ρ dependent mechanism of transcriptional termination, a string of As repeat is not
present in template strand. In this case when stem loop structure is formed, a protein
ρ (rho) recognizes and binds to rut (rho utilization) element that is a CA rich
sequence. Rhoprotein has two structural domains, one of which binds with ATP that
helps the rho protein for translocation along the RNA molecule and second domain
binds to RNA molecule. The hydrolysis of ATP facilitates the migration of ρ protein
along RNA strand in 5´---->3´ direction. This protein breaks the association between
DNA:RNA duplex with its activity like helicases and as a result, RNA transcript is
released and transcription termination process is completed.
In prokaryotes, the genes of rRNA contain another element in addition to -10 box
and -35 box in their promoter regions that is called UP (upstream element). This UP
element is ~20 bps upstream to -35 box (Fig 4.5a) and in rRNA synthesis, RNA
polymerase α subunit recognizes UP element and σ subunit recognizes -10 box and
-35 box promoter regions. There are also some genes in prokaryotes which neither
have UP element nor -35 box in their promoter region. In these genes, an additional
short extended region is present just upstream the -10box, and is known as extended
-10 element (Fig 4.5b) (Tamarin 2001; Lewin 2004; Turner et al. 2005).
42 Chapter 4

Fig. 4.5 In prokaryotes some genes have additional elements in their promoter
regions (a) rRNA genes have additional UP element in their promoter region that is
recognized by α subunit of RNA polymerase (b) Some genes have neither -35 box
nor UP elements, rather an extended -10 element within -10 box is present in their
promoter region (Conceived from Watson et al. 2004).

4.4 Post Transcriptional modifications in prokaryotes

There is no post transcriptional modification event for mRNA of prokaryotes while
rRNA and tRNA of prokaryotes undergo post transcriptional processing. Three types
of rRNA (23S, 16S and 5S) are present in prokaryotes, as parts of ribosome.
Prokaryotic ribosome (70S) has two subunits; 30S small subunit and 50S large
subunit (Fig 4.6). The 23S and 5S rRNA along with 31 different kinds of proteins
form 50S subunit and 16S rRNA along with 21 different proteins make 30S subunit
of ribosome in prokaryotes.
Genes for 23S, 16S and 5S rRNAs are present as transcriptional units called rrn
operon. Seven copies of rRNA genes are present in E. coli. Each copy of rRNA gene
contains sequences for 16S, 23S and 5S rRNAs as well tRNA gene too (in some
cases). In some rRNA transcriptional units (rrn), tRNA genes are present between
16S and 23S rRNA sequences, whereas in some instances gene for tRNA is present
at the end of 5S rRNA sequence (Fig 4.7) (Lodish et al. 2012; Nelson and Cox 2012).
Mechanism of Transcription in Prokaryotes 43

Fig. 4.6 Prokaryotic ribosome and its subunits

(a)

(b)
Fig. 4.7 Transcriptional unit of rRNA genes or rrn operon (a) In some transcriptional
unit of rRNAs, tRNA gene is present at the end 5S rRNA (b) whereas in some
transcriptional unit of rRNAs, tRNA gene is present between 16S rRNA and 23S
rRNA sequences (Taken after modification from Nelson and Cox 2012)

The rRNA genes in a transcriptional unit are transcribed into 30S pre-rRNA that
contains 16S, 23S, 5S rRNAs along with tRNA and its methylation at specific bases
takes place afterwards. This pre-rRNAs is then processed and each rRNAs along with
tRNA is separated after the cleavage with RNaseIII, RNaseP and RNaseE enzymes.
The further processing at 5΄ and 3́ end of each rRNA by other ribonucleases (M23,
M16 and M5) is carried out; thus rRNAs are matured (Fig 4.8), and interact with
other proteins to form ribosome (Watson et al. 2004; Turner et al. 2005).
44 Chapter 4

Fig. 4.8 Transcription and processing of rRNA transcriptional unit (rrn) of E.coli
and formation of mature rRNAs (Modified from Nelson and Cox 2012)

4.5 Messenger RNA (mRNA) degradation in

prokaryotes
The population of mRNA in a cell is mainly dependent on the rate of biogenesis of
mRNA and its degradation rate. In prokaryotes, a multi-nucleolytic protein complex;
degradosome, decays RNA and acts as RNA decay machine that also maintains RNA
turnover rate. This prokaryotic RNA decay machine has four major parts; RNaseE,
PNPase (polynucleotide phosphorylase), enolase and helicase (Rh1B) (Fig 4.9)
(Coburn and Mackie 1999; Carpousis et al. 1999; Leroy et al. 2002). RNase E is the
Mechanism of Transcription in Prokaryotes 45

major and pivotal part of degradosome, having a key role in the decay of specific
transcript It has three domains; N-terminus catalytic domain, the arginine-rich RNA-
binding domain (RBD) and C-terminus domain/noncatalytic region. RNase E, being
a scaffold protein interacts with other proteins of this complex as well as with RNA
at its C terminus domain (Carpousis 2002;Wang et al. 2002; Burger 2010). The
arginine-rich RNA-binding domain (RBD) is a site of RNA:protein interaction, while
N terminus domain of RNase E joins the degradosome to cytoplasmic membrane.
The helicase component of degradosome has a role in strands separation whereas
cutting of mRNA into short pieces is achieved through RNaseE and the degradation
of small RNA pieces into nucleotides is accomplished by PNPase (Baker and Mackie
2003; Carpousis 2007; Carpousis et al. 2009).

Fig. 4.9 Degradosome.A prokaryotic RNA decaying machine (Redrawn after

modification from Kaberdin et al. 2011)

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