PROKARYOTIC MESSENGER RNA

CONTENTS
Page No

1. Introduction………………………………………………………………… 2,3

2.Structure of Messenger RNA – mRNA……………………………………. 3,4

3. Size of Messenger RNA – mRNA………………………………………….. 4

4.Synthesis of Messenger RNA – mRNA…………………………………….. 4,5

5.Stability of Messenger RNA – mRNA……………………………………… 5

6. Very stable prokaryotic messenger RNA in chromosomeless Escherichia coli

minicells………………………………………………………………………… 5,6

7.Prokaryotic translation…………………………………………………………………… 6,7,8

8. Control of Escherichia coli Messenger RNA Degradation…………….

………………………………………………………………………… 8,9,10,11,12,13,14,15

9.Difference Between Prokaryotes and Eukaryotes mRNA…………………. 15,16

10.References…………………………………………………………………… 17

1
 PROKARYOTIC MESSENGER RNA

1. Introduction:-
Messenger ribonucleic acid (mRNA):-
is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is
transcribed from a DNA template, and carries coding information to the sites of protein
synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of
amino acids: a protein. In mRNA as in DNA, genetic information is encoded in the
sequence of nucleotides arranged into codons consisting of three bases each. Each codon
encodes for a specific amino acid, except the stop codons that terminate protein synthesis.
This process requires two other types of RNA: transfer RNA (tRNA) mediates
recognition of the codon and provides the corresponding amino acid, while ribosomal
RNA (rRNA) is the central component of the ribosome's protein manufacturing
machinery.[1]

2
 PROKARYOTIC MESSENGER RNA

Messenger ribonucleic acid (mRNA), like DNA, carries unique codes in different series
of patterns that relay messages to structures within the cell. Messenger RNA, unlike
DNA, is specifically responsible for encoding information and carrying it to the site of
protein synthesis, the ribosomes, where proteins are utilized within a cell as they are
translated from nucleotides to proteins that are able to produce energy for the cell to use
to carry out its daily functions.[4]
Jacob and Monod (1961) proposed the name messenger RNA for the RNA carrying
information for protein synthesis from the DNA (genes) to the sites of protein formation
(ribosomes). It consists of only 3 to 5% of the total cellular RNA.
Messenger RNA (mRNA) carries information about a protein sequence to the ribosomes,
the protein synthesis factories in the cell. It is coded so that every three nucleotides (a
codon) correspond to one amino acid. In eukaryotic cells, once precursor mRNA (pre-
mRNA) has been transcribed from DNA, it is processed to mature mRNA. This removes
its introns—non-coding sections of the pre-mRNA. The mRNA is then exported from the
nucleus to the cytoplasm, where it is bound to ribosomes and translated into its
corresponding protein form with the help of tRNA. In prokaryotic cells, which do not
have nucleus and cytoplasm compartments, mRNA can bind to ribosomes while it is
being transcribed from DNA. After a certain amount of time the message degrades into
its component nucleotides with the assistance of ribonucleases.[2]

2.Structure of Messenger RNA – mRNA:-

Messenger RNA is always single stranded. It contains mostly the bases adenine, guanine,
cytosine and uracil. There are few unusual substituted bases. Although there is a certain
amount of random coiling in extracted mRNA, there is no base pairing. In fact base
pairing in the mRNA strand destroys its biological activity.
Since mRNA is transcribed on DNA (genes), its base sequence is complementary to that
of the segment of DNA on which it is transcribed. This has been demonstrated by
hybridization experiments in which artificial RNADNA double strands are produced.
Hydrization takes place only if the DNA and RNA strands are complementary.
Usually each gene transcribes its own mRNA. Therefore, there are approximately as
many types of mRNA molecules as there are genes. There may be 1,000 to 10.000
different species of mRNA in a cell. These mRNA types differ only in the sequence of
their bases and in length.
When one gene (cistron) codes for a single mRNA strand the mRNA is said to be
monocistronic. In many cases, however, several adjacent cistrons may transcribe an
mRNA molecule, which is then said to be polycistronic or polygenic.
The mRNA molecule has the following structural features:
1. Cap. At the 5' end of the mRNA molecule in most eukaryote cells and animal
virus molecules is found a 'cap'. This is blocked methylated structure, m7Gpp
Nmp Np or m7Gpp Nmp Nmp Np. where: N = any of the four nucleotides and
Nmp = 20 methyl ribose. The rate of protein synthesis depends upon the presence
of the cap. Without the cap mRNA molecules bind very poorly to the ribosomes.
2. Noncoding region 1 (NC1). The cap is followed by a region of 10 to 100
nucleotides. This region is rich in A and U residues, and does not translate
protein.

3
 PROKARYOTIC MESSENGER RNA

3. The initiation codon is A UG in both prokaryotes and eukaryotes.

4. The coding region consists of about 1,500 nucleotides on the average and
translates protein. [3]

3. Size of Messenger RNA - mRNA :-

The molecular weight of an average sized mRNA molecule is about 500,000, and its
sedimentation coefficient is 8S. It should be noted however, that mRNA varies greatly in
length and molecular weight. Since most proteins contain at least a hundred amino acid
residues, mRNA must have at least 100 X 3= 300 nucleotides on the basis of the triplet
code.
In E. coli the average mRNA strand has 900 to 1,500 nucleotide units which would code
polypeptide chains of 300-500 amino acids. Molecules containing 12,000 nucleotide units
are also known.[3]

4.Synthesis of Messenger RNA - mRNA :–

Messenger RNA is transcribed on a DNA strand through the enzymatic action of RNA
polymerase. Synthesis begins at the 5' end and proceeds to the 3' end. RNA polymerase
attaches to DNA at the initiator site and opens up a short segment of the DNA double
helix.
There is complementary base pairing between the free bases of one of the DNA strands
and ribonucleotides, resulting in the formation of an mRNA strand. The RNA polymerase
moves along the DNA template, and the growing mRNA strand is peeled off till a
termination site is reached.

4
 PROKARYOTIC MESSENGER RNA

Hydrogen bonding then again takes place between the complementary bases of the two
DNA strands. Synthesis of mRNA is similar to replication of DNA except for the
following differences: (1) ribose nucleotides are used instead of deoxyribose nucleotides;
(2) adenine pairs with uracil instead of thymine, and (3) only one strand of DNA
transcribes the mRNA strand.
The transcribed strand undergoes processing before it becomes mRNA. During
processing elements that are no longer of any use are removed and elements thus make
the RNA functionally more effective are added. Prokaryotic mRNAs normally undergo
very little processing. In bacterial cells there is a very short time interval between
transcription and translation.
A large proportion of the mRNA may be translated before transcription is completed. In
some cases degradation of mRNA may begin even before completion of transcription.
Methylation and polyadenylation which occur to a considerable extent in eukaryotes are
not prevalent in bacterial cells. Nevertheless a certain amount of processing does occur.
In the T7 phage five contiguous genes transcribe 'early RNA' which is cut during
transcription to five individual mRNAs by RNase III. In eukaryotes the transcribed RNA
molecules are larger than mature mRNA by 20% in the slime mould Dictyostelium and
about X 4 to X 5 in mammalian cells. The large RNA molecules containing pre-mRNA
transcripts are known as heterogeneous nuclear RNAs (hnRNA).[3]

5.Stability of Messenger RNA – mRNA: -

The cell does not contain large quantities of mRNA. This is because mRNA, unlike other
RNAs is constantly undergoing breakdown. It is broken down to its constituent
ribonucleotides by ribonucleases.
In bacteria, mRNA may be so short lived that while one end is translating protein the
other end may be undergoing breakdown. In E. coli the average half-life of some mRNAs
is about two minutes.Eukaryotic cells, on the other hand, contain metabolically stable
mRNAs.
Mammalian reticulocytes are immature RBC which have lost their nuclei but have
retained ribosomes. These cells can synthesize haemoglobin for many hours and even
days by utilizing the mRNA transcribed by the nuclei when they were present.[3]

6. Very stable prokaryotic messenger RNA in chromosomeless

Escherichia coli minicells.
E. coli minicells lack DNA, yet they make protein, the synthesis of which is sensitive to
chloramphenicol but insensitive to rifamycin. This protein is coded for by very stable
cellular mRNA with an estimated half-life of 40-80 min. In an R factor-containing
minicell, two very different species of mRNA are observed: (i) R factor-specific mRNA
with a short half-life whose synthesis is rifamycin-sensitive and (ii) cellular mRNA with
a long half-life whose synthesis is rifamycin-insensitive. These findings indicate that
minicells contain normal degradative mechanisms for mRNA and point out the existence
of a unique class of very stable cellular mRNA. Greater than 80% of the rifamycin-
insensitive protein synthesized goes into the outer minicell membrane. Relatively stable
mRNA, half-life 5.5-11.5 min, for outer membrane protein in whole cells has been
reported [Hirashima et al. (1973) J. Mol. Biol. 79, 373-389]. The stability of minicell
mRNA is significantly greater. This and other observations suggest that there are two

5
 PROKARYOTIC MESSENGER RNA

functional species of mRNA for outer membrane protein perhaps in different sites in the
cell. Furthermore, these studies suggest that a class of cellular proteins is synthesized in
bacteria without concomitant transcription and in the absence of association with
chromosomal DNA.[6]

7.Prokaryotic translation:-
is the process by which messenger RNA is translated into proteins in prokaryotes.[5]

7.1 Initiation

of translation in prokaryotes involves the assembly of the components of the translation

system which are: the two ribosomal subunits (small and large), the mRNA to be
translated, the first (formyl) aminoacyl tRNA (the tRNA charged with the first amino
acid), GTP (as a source of energy), and three initiation factors (IF1, IF2, and IF3) which
help the assembly of the initiation complex.The ribosome has three sites: the A site, the P
site, and the E site. The A site is the point of entry for the aminoacyl tRNA (except for the
first aminoacyl tRNA, fMet-tRNAfMet, which enters at the P site). The P site is where the
peptidyl tRNA is formed in the ribosome. And the E site which is the exit site of the now
uncharged tRNA after it gives its amino acid to the growing peptide chain.

The process of initiation of translation in prokaryotes.

7.2 Elongation

6
 PROKARYOTIC MESSENGER RNA

Elongation of the polypeptide chain involves addition of amino acids to the carboxyl end
of the growing chain. The growing protein exits the ribosome through the polypeptide
exit tunnel in the large subunit.Elongation starts when the fmet-tRNA enters the P site,
causing a conformational change which opens the A site for the new aminoacyl-tRNA to
bind. This binding is facilitated by elongation factor-Tu (EF-Tu), a small GTPase. Now
the P site contains the beginning of the peptide chain of the protein to be encoded and the
A site has the next amino acid to be added to the peptide chain. The growing polypeptide
connected to the tRNA in the P site is detached from the tRNA in the P site and a peptide
bond is formed between the last amino acids of the polypeptide and the amino acid still
attached to the tRNA in the A site. This process, known as peptide bond formation, is
catalyzed by a ribozyme, peptidyltransferase, an activity intrinsic to the 23S ribosomal
RNA in the 50S ribosomal subunit. Now, the A site has the newly formed peptide, while
the P site has an uncharged tRNA (tRNA with no amino acids). In the final stage of
elongation, translocation, the ribosome moves 3 nucleotides towards the 3'end of mRNA.
Since tRNAs are linked to mRNA by codon-anticodon base-pairing, tRNAs move
relative to the ribosome taking the nascent polypeptide from the A site to the P site and
moving the uncharged tRNA to the E exit site. This process is catalyzed by elongation
factorGEF-G).
The ribosome continues to translate the remaining codons on the mRNA as more
aminoacyl-tRNA bind to the A site, until the ribosome reaches a stop codon on
mRNA(UAA, UGA, or UAG).

7.3Termination

Termination occurs when one of the three termination codons moves into the A site.
These codons are not recognized by any tRNAs. Instead, they are recognized by proteins
called release factors, namely RF1 (recognizing the UAA and UAG stop codons) or RF2
(recognizing the UAA and UGA stop codons). These factors trigger the hydrolysis of the
ester bond in peptidyl-tRNA and the release of the newly synthesized protein from the
ribosome. A third release factor RF-3 catalyzes the release of RF-1 and RF-2 at the end
of the termination process.

7.4 Recycling

The post-termination complex formed by the end of the termination step consists of
mRNA with the termination codon at the A-site, an uncharged tRNA in the P site, and the
intact 70S ribosome. Ribosome recycling step is responsible for the disassembly of the
post-termination ribosomal complex. Once the nascent protein is released in termination,
Ribosome Recycling Factor and Elongation Factor G (EF-G) function to release mRNA
and tRNAs from ribosomes and dissociate the 70S ribosome into the 30S and 50S
subunits. IF3 then replaces the deacylated tRNA releasing the mRNA. All translational
components are now free for additional rounds of translation.

7.5 Polysomes

7
 PROKARYOTIC MESSENGER RNA

Translation is carried out by more than one ribosome simultaneously. Because of the
relatively large size of ribosomes, they can only attach to sites on mRNA 35 nucleotides
apart. The complex of one mRNA and a number of ribosomes is called a polysome or
polyribosome.] Effect of antibiotics

7.6 Effect of antibiotics

Several antibiotics exert their action by targeting the translation process in bacteria. They
exploit the differences between prokaryotic and eukaryotic translation mechanisms to
selectively inhibit protein synthesis in bacteria without affecting the host.[5]

8. Control of Escherichia coli Messenger RNA Degradation:-

8.1. Ribonucleases
Ribonucleases (RNases) are a class of enzymes that are responsible for RNA degradation
and processing. These enzymes are classified as endo- and exoribonucleases (Table
1). In addition, an RNase with unique exoribonucleolytic activity has recently been
described in B. subtilis [26, 27]. At least two ribonucleases are components of a
holoenzyme complex, the RNA degradosome, which catalyzes bulk E. coli mRNA
degradation. In addition to the RNA degradosome, E. coli produces other endo- and
exoribonucleases, many of which are largely considered to be involved in rRNA and
tRNA maturation rather than bulk mRNA decay. A subset of these enzymes contribute to
the decay of individual mRNA species, whereas others have not yet, or have only
circumstantially, been linked to mRNA degradation. It is likely that as each of these
ribonucleases becomes better characterized, many will be found to contribute to mRNA
decay. In the pages that follow, we describe components of the E. coli RNA degradosome
and other RNases that mediate processing/degradation of targeted bacterial mRNA
species. Moreover, we discuss several mechanisms by which the activity of the
degradosome and other bacterial RNases is modulated as a means of regulating mRNA
levels during cellular proliferation and stress conditions. [7]

8.1.1. The Degradosome

The E. coli degradosome is composed of at least four proteins: ribonuclease E (RNase E),
polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and the glycolytic enzyme
enolase . As shown in Figure 1, a model for mRNA decay has been proposed in which
the degradosome loads and scans an mRNA molecule for an RNase E cleavage site, A/U-
rich sequences usually proceeded by a stem-loop structure in monophosphorylated
transcripts, in the direction . Once this site is encountered, RNase E catalyzes an
initial endoribonucleolytic event and then continues to cleave the transcript at additional
downstream target sites. Fragmentation products are subsequently degraded by the
exoribonuclease, PNPase . RhlB RNA helicase-mediated removal of mRNA secondary
structures is thought to facilitate PNPase degradation, whereas enolase may participate in
bulk degradation of metabolic enzyme transcripts .

8
 PROKARYOTIC MESSENGER RNA

Degradosome-mediated RNA decay. The E. coli degradosome is composed of at least

four subunits: RNase E, PNPase, RhlB helicase, and enolase. The initial RNA cleavage
event is catalyzed by the endoribonuclease RNase E (large cut-out circle) which
loads onto a transcript and scans for downstream cleavage sites: A/U rich regions
proceeded by stem-loop structures in monophosphorylated transcripts. The
exoribonuclease PNPase (small cut-out circle) catalyzes cleavage of RNase E-generated
decay intermediates. Otherwise inhibitory secondary structures to PNPase-mediated
degradation are resolved by RhlB helicase (cross). The role of enolase (hexagon) in
mRNA decay is not well characterized.Components of the degradosome are localized to
the cell membrane and are organized into helical filaments that coil around the length of
the cell [34–36]. This organization may provide a means for the apparatus to interact with
other cell membrane-associated macromolecular complexes [35, 36]. Indeed, the
degradosome appears to be a dynamic organelle; during cold shock conditions, the
complex’s RNA helicase is replaced by an alternative cold shock RNA helicase, CsdA
[37]. The cold shock protein CspE also interacts with degradosome-associated
ribonucleases [38]. Further, the heat shock proteins GroEL and DnaK have been shown
to be associated with the degradosome [39]. It remains to be seen whether these auxiliary
factors affect global mRNA decay. Rather, it seems likely that they may redirect the
efficiency with which the degradosome catalyzes turnover of individual or subsets of
mRNA species. This would provide an efficient means of modulating protein production
in a manner that allows cells to quickly adapt to otherwise deleterious conditions. It is
very likely that as the field matures, additional degradosome auxiliary factors will be
identified and characterized. As a first step toward understanding how the holoenzyme’s
function can be altered in response to endogenous and exogenous cues, one must first
appreciate the components of the “native” RNA degradosome complex.

RNase E [rne; 118 kilodalton (kDa)] is an essential endoribonuclease that organizes other
components of the E. coli degradosome and initiates bulk RNA decay. The C-terminal
region of RNase E acts, in part, as a scaffold for assembly of the other major
degradosome components] The protein’s internal domain is required for cell membrane
association, whereas its N-terminus is required for cell viability and RNA cleavage [31,
41, 42]. In addition to its role in mediating bulk mRNA degradation, RNase E is involved
in the maturation of both ribosomal and transfer RNA molecules

Because RNase E is responsible for many RNA decay and maturation processes, it stands
to reason that it must be tightly regulated. Indeed, RNase E autoregulates itself by
controlling the cleavage of its cognate mRNA. When RNase E activity is low or when
substrate transcripts reach high levels, the rate of rne cleavage is reduced which results in
increased RNase E production. As substrate molecules are depleted, rne mRNA
degradation and protein production return to basal levels. As discussed further below,
trans-acting factors such as noncoding RNAs, RNA binding proteins, and the translation
apparatus frequently indirectly affect RNase E function by altering a target transcript’s
accessibility to the enzyme.

9
 PROKARYOTIC MESSENGER RNA

During normal laboratory growth conditions, polynucleotide phosphorylase (PNPase;

pnp; 80 kDa) functions as a nonessential, exoribonuclease component of the
degradosome]Although other cellular exoribonucleases can rescue a loss of PNPase
activity, they do so with reduced efficiency; pnp-mutants produce transcripts with mildly
increased steady-state levels. As opposed to normal growth conditions, PNPase is
essential for survival at low temperatures (<20 ). Following cold acclimation, the
enzyme is required for degradation of low temperature stabilized transcripts whose
accumulation would otherwise be lethal. The temperature mediated change in cellular
PNPase dependence suggests that ribonuclease functions/importance changes in response
to internal and/or external stimuli. As the field matures, it is likely that this phenomenon
will be observed for additional RNases. In addition to its role in degradosome-mediated
RNA degradation and cold shock adaptation, PNPase may also participate in
polyadenylation of mRNA .

Although not ribonucleases, RhlB helicase and enolase are integral members of the E.
coli degradosome. RhlB (rhlB; 47 kDa) is a DEAD box RNA helicase that unwinds RNA
secondary structures via energy generated by ATP hydrolysis. Presumably, RhlB
facilitates PNPase-mediated digestion of RNase E-generated fragments. The glycolytic
enzyme enolase (eno; 46 kDa) is an abundant E. coli protein; ~10% of all cellular enolase
is associated with the degradosome. Although the function of enolase as part of the
degradosome has not been elucidated, some studies have indicated a possible role for
enolase in bulk mRNA turnover of some metabolic enzymes. [7]

8.1.2. Endoribonucleases

RNase III (rnc; 26 kDa) is an endoribonuclease that cleaves double-stranded RNA.

Although the enzyme is best known for its role in rRNA maturation, RNase III also
regulates the mRNA decay of a subset of RNA species, including pnp, which contain a
stem loop structure. Other noted RNase III substrates include the intergenic regions of
rplL-rpoB]rpsO-pnp dicA-dicF-dicB , and metY-nusA transcripts. In addition to its role in
degrading target RNA molecules, RNase III has been shown to bind the untranslated
region (UTR) of bacteriophage cIII transcripts. Binding alters the mRNA conformation
and alleviates an otherwise translation-inhibitory structure. Thus, RNase III has at least
two post-transcriptional regulatory mechanisms which are facilitated by its RNA binding
and/or RNA degradation activities.RNase P is a holoenzyme consisting of a ribozyme
(rnpB; 377 nucleotides) and at least one protein subunit, RnpA (rnpA; 14 kDa). The
major function of the ribonucleoprotein complex has been considered to be catalyzing
cleavage of the leader sequence of precursor tRNAs. It is well established that the
ribozyme is the catalytic unit, whereas the protein component aids in substrate
recognition. Interestingly, although both the RNase P ribozyme and protein subunits are
essential in vivo, the protein subunit is not required for in vitro precursor tRNA
processing .RNase P has also been implicated in the cleavage of intergenic regions of
polycistronic mRNA molecules and the degradation of guide RNAs. Thus, RNase P may
facilitate both tRNA maturation and degradation of subsets of mRNA species during
distinct conditions. Indeed, RNase P activity is regulated in response to nutrient
limitation.Two ribonuclease H genes exist within E. coli. Although they have similar

10
 PROKARYOTIC MESSENGER RNA

functions, they share limited sequence similarity. RNase HI (rnhA; 18 kDa) was the first
to be identified. The enzyme degrades the RNA component of RNA/DNA duplexes. A
precise physiological role has not been determined for RNase HI. However, potential
functions have been proposed including the removal Okazaki fragment primers as well as
primers at sites other than the vegetative origin of replication. The second E. coli
ribonuclease H gene, RNase HII (rnhB; 23 kDa), also degrades the RNA component of
RNA/DNA hybrid molecules. Like RNase HI, the enzyme’s biological function is
unknown. RNase G (rng/cafA; 55 kDa) was initially termed CafA because it was first
determined to be involved in cell division and the formation of cytoplasmic axial
filaments. CafA shares N-terminal amino acid homology with RNase E, thus, it was not
surprising when the protein was found to exhibit endoribonuclease activity with
specificity to A/U-rich sequences and was subsequently renamed RNase G. Despite the
similarities to RNase E, RNase G is not essential and is not responsible for bulk E. coli
mRNA decay. Nonetheless, RNase G appears to affect the mRNA turnover of at least two
transcripts: fermentative aldehyde dehydrogenase (adhE) and enolase (eno). RNase BN/Z
(elaC; 33 kDa) is a nonessential endoribonuclease in E. coli. In other organisms, the
enzyme cleaves CCA-less tRNA molecules endonucleolytically. However, all E. coli
tRNAs have chromosomally encoded CCAs and thus are not cleaved by RNase Z.
Nonetheless, RNase Z is able to mature tRNAs in the absence of the other 4 tRNA
maturation exoribonucleases (see below). Furthermore, the steady-state levels of over 150
transcripts increased in the absence of RNase Z including rpsT, cspE, htpG, glpQ, and
adhE.RNase LS (rnlA; 40 kDa) was initially identified as a regulator of bacteriophage T4
late gene silencing, which is responsible for degrading the corresponding transcripts.
RNase LS also moderately affects the turnover of several E. coli transcripts and
profoundly affects the stability of bla and an accumulated fragment of 23S rRNA. It has
been suggested that RNase LS exists in a multiprotein 1000 kDa complex which indicates
that the activity of the enzyme is dependent on interactions with other proteins. RNase I
(rna; 29 kDa) is a nonessential and nonspecific endoribonuclease that resides in the E.
coli periplasmic space which provides a unique mechanism by which the enzyme’s
activity can be tightly regulated. During nonstress conditions, the cytoplasmic
concentration of RNase I is presumably low. However, during stress-induced
transcriptional arrest, RNase I leaks from the periplasmic space into the cytoplasm where
it rapidly degrades rRNA and tRNA. Variants of RNase I (RNase M and RNase I*) are
present in E. coli and have been shown to degrade mRNA. Expression of chromosomally
encoded toxin-antitoxin systems enables cells to rapidly shut down cellular processes in
response to changes in growth conditions. These bicomponent systems consist of a short-
lived antitoxin and a stable toxin. Under normal growth conditions, the antitoxin silences
the toxin. However, in stress-inducing environments when cellular processes are
downregulated, the antitoxin is rapidly degraded resulting in derepression of the toxin.
Studies on the targets of the toxin components of these systems have indicated some
function as ribonucleases. There are two classes of toxin-mediated ribonucleases in E.
coli: (1) toxins that cleave mRNA molecules present in ribosomes which include RelE
and YoeB and (2) toxins that cleave mRNAs independent of translation which includes
MazF and ChpBK. Both classes of toxin-mediated ribonucleases inhibit translation and,
consequently, protein production by degrading target transcripts.[7]

11
 PROKARYOTIC MESSENGER RNA

8.1.Exoribonucleases
Seven E. coli exoribonucleases have been identified. Of these PNPase, RNase II, RNase
R, and Oligo-RNase are established to affect mRNA degradation and, consequently,
protein production. The remaining exoribonucleases RNase PH, RNase D , and RNase T.
are believed to function primarily as tRNA maturation enzymes; none have been
identified to modulate mRNA turnover. However, it is important to recognize that formal
studies designed to globally measure what effect, if any, these enzymes have on mRNA
turnover have not been described. Thus, one cannot rule out the possibility that they may
also affect mRNA degradation, and until proven otherwise, it is possible that virtually
any defined ribonuclease may play a role in the mRNA turnover of individual or subsets
of bacterial transcripts.RNase R (rnr; 95 kDa) is a processive exoribonuclease that
cleaves structured polyadenylated [poly(A)] mRNA, tRNA, and rRNAs in vitro. Thus, it
is thought that the in vivo role of RNase R is to degrade highly structured RNA
molecules, such as those containing repetitive extragenic palindromic sequences which
are associated with stable stem-loops. RNase R can degrade these secondary structures in
the absence of an RNA helicase provided there is a single-stranded region, such as a
poly(A) tail, available for the enzyme to bind and initiate decay. RNase R activity
increases in response to several stress conditions including entry into stationary phase,
starvation, and cold shock . During these conditions, RNase R has been proposed to
catalyze degradation of structured RNA molecules when protein production needs to be
stalled. RNase II (rnb; 72 kDa) is a processive exoribonuclease that accounts for
~90% of all exoribonucleolytic activity of poly(A) RNA. The enzyme processes the tail
of immature tRNA. It also regulates the stability of mRNA by removing poly(A) tails
which makes them less accessible to the degradosome. Oligo-RNase (orn; 38 kDa) is an
essential, processive exoribonuclease which, as the name implies, cleaves short
oligoribonucleotides. The enzyme copurifies with PNPase suggesting that it may catalyze
digestion of oligoribonucleotide intermediates generated during PNPase-mediated mRNA
decay.[7]

8.2. RNA Binding Proteins

Another major class of mRNA turnover regulatory molecules includes RNA binding
proteins. As shown in Figure 2, their binding frequently stabilizes or destabilizes mRNA
species by affecting the transcripts susceptibility to ribonuclease digestion. Examples of
two well-characterized RNA binding proteins are discussed below.The Host factor I
protein (Hfq; 11 kDa) is an RNA binding protein that affects mRNA stability by
facilitating base pairing between sRNAs (described below) and their mRNA targets. This,
in turn, can increase or decrease a transcript’s accessibility to ribonucleases. For example,
in rapidly growing cells, outer membrane protein A (ompA) mRNA is stabilized by
elements in its UTR .However, upon entry into stationary phase, the noncoding sRNA,

12
 PROKARYOTIC MESSENGER RNA

MicA/SraD, is induced and binds ompA mRNA. Hfq binds both ompA and the sRNA in
vitro and presumably facilitates base pairing between these RNAs in vivo. Pairing
inhibits ribosome binding and promotes RNase E-dependent degradation. Additional
examples of Hfq-mediated sRNA:mRNA pairing are discussed below. In addition to
catalyzing RNA degradation, Hfq has been shown to stabilize DsrA, RyhB, and OxyS
transcripts, all of which are well-studied sRNAs. In these cases, Hfq binding overlaps
with RNase E cleavage sites thereby reducing the transcripts’ accessibility to
ribonuclease attack. In addition to its role in facilitating sRNA:mRNA pairing, Hfq
stabilizes transcripts by enhancing the PAP I-mediated elongation of poly(A) tails in vivo
and in vitro. The carbon storage regulator protein (CsrA; 7 kDa) is a negative regulator of
postexponentially induced metabolic pathways; a positive regulator of glycolysis, acetate
metabolism, and motility; and a repressor of biofilm formation in E. coli . The protein’s
regulatory effects are, in part, due to its ability to modulate the mRNA turnover of target
transcripts. This has been best characterized for the polycistronic glycogen biosynthesis
transcript, glgCAP. During exponential phase growth when nutrient sources are readily
available, CsrA binds to the UTR of glgCAP mRNA which, in turn, inhibits ribosome
loading and promotes transcript degradation. During stationary phase growth, glycogen
biosynthesis is upregulated, in part, because CsrA no longer efficiently binds glgCAP
transcripts. Rather, the protein becomes predominantly sequestered into a
ribonucleoprotein complex comprised of 18 CsrA subunits and one small RNA,
CsrB.Although not as extensively characterized, CsrA also seems to regulate the
transcript stability and, consequently, protein production of several virulence factors.
However, the effects of CsrA binding appear to be transcript specific. For instance, CsrA
decreases the half-life of pgaABCD which prevents PGA (poly-beta-1,6-N-acetyl-d-
glucosamine) production; a cell surface polysaccharide that promotes biofilm formation.
Thus, CsrA’s mechanism of action may be similar to its role in glgCAP regulation;
binding mayinhibit translation initiation and increase ribonuclease degradation.
Conversely, CsrA stabilizes the flagellar transcriptional activator genes flhDC,
presumably by binding to the transcript. Thus, CsrA promotes motility either by acting as
an activator of translation or by protecting the transcript from ribonuclease digestion. As
described above, Hfq and CsrA are two well-characterized E. coli RNA binding proteins
that influence protein production by altering mRNA stability. In addition, the histone-like
protein H-NS regulates mRNA stability by binding to target transcripts. Other H-NS like
proteins regulate mRNA stability as well. Further studies will likely identify additional
RNA binding proteins that regulate gene expression by altering mRNA stability.[7]

8.3. Small RNAs

The third class of regulatory molecules discussed here is small noncoding RNAs
(sRNAs). More than 80 sRNAs have been identified in E. coli; many of these are
components of stress responsive regulons. sRNAs typically do not have a discernable
open reading frame encoded in their sequence, thus the RNA molecule rather than a
protein product is thought to affect gene expression. As described by the Aiba laboratory,
the regulatory effects of sRNAs are mediated largely by their binding to mRNA and
affecting translation which, in turn, mediates turnover of target transcripts Other sRNAs
such as the ribozyme rnpB, tmRNA, and 4.5S regulate gene expression through entirely

13
 PROKARYOTIC MESSENGER RNA

different processes. rnpB processes tRNA molecules and thus affects translation 4.5S is
part of the signal recognition particle ribonucleoprotein complex that targets membrane
and secreted proteins for translocation during translation, and tmRNA is a quality control
regulator that rescues stalled ribosomes and facilitates the elimination of proteins whose
translation has been prematurely terminate. In the following section, we will overview
sRNAs that function as antisense regulatory molecules to influence transcript stability.
For more detailed information regarding the identification and other mechanisms of
sRNA regulation, we refer the reader to several excellent reviews .One of the best-studied
sRNAs is DsrA which affects the mRNA turnover of at least two target transcripts: hns
and rpoS. As in the case of the RNA binding protein CsrA, DsrA catalyzes digestion of
certain transcripts but stabilizes others. For example, under normal growth conditions, the
stationary phase RNA polymerase sigma factor, rpoS, transcript is destabilized by the
formation of a stable hairpin in its UTR. Doing so sequesters the ribosome binding site
and promotes RNase III-mediated transcript degradation.However, during nonoptimal
growth conditions, Hfq catalyzes antisense base pairing between DsrA and rpoS enabling
efficient translation which stabilizes the message and results in increased protein
production. In contrast, DsrA base pairing with the histone-like protein transcript hns
inhibits ribosome entry which destabilizes the message resulting in decreased H-NS
abundance .The ferric uptake regulator, Fur, has classically been considered a repressor
protein but also indirectly activates gene expression in response to iron availability via an
sRNA. When is abundant, Fur becomes activated and inhibits expression of genes
involved in various iron acquisition systems. Other genes including those involved in iron
storage and intracellular usage are activated by Fur during these same conditions. Massé
and Gottesman have shown that Fur-mediated gene activation is indirect and involves the
sRNA RyhB. In that study, it was found that Fur represses RyhB synthesis when iron is
abundant which, in turn, induces the expression of proteins that bind intracellular iron.
However, when iron availability is limited, Fur becomes inactivated resulting in RyhB
upregulation and repression of target genes. RyhB represses gene expression by binding
to target transcripts in an Hfq-dependent manner which facilitates RNase E-mediated
mRNA degradation. Other examples of stress-induced sRNAs include OxyS (oxidative
stress;), OmrA/B (osmotic shock;), RprA (cell surface stress;), MicA/SraD (stationary
phase; ), MicF (oxidative/antibiotic stress;), SgrS (glucose phosphate accumulation;), and
Spot 42 (glucose limitation). We do not intend to give the impression that the three
classes of molecules discussed above, RNases, RNA binding proteins, and sRNAs, are
the sole mediators of bacterial mRNA turnover. In fact, Deborah Steege published an
excellent review highlighting the identification, characterization, and cellular role of
polyadenylation in bacteria. Poly(A) polymerase I (pcnB; 53 kDa) is responsible for
adding 10–40 nt poly(A) tails to bacterial RNA species. Although polyadenylated
transcripts account for only 0.01–2% of the total cellular mRNA content, polyadenylation
plays a significant role in regulating the stability of target transcripts. Indeed, E. coli
polyadenylated mRNA molecules are rapidly degraded by the exoribonucleases
RNase II and PNPase. It is believed that poly(A) tails provide a single-stranded extension
region upon which these RNases can bind and initiate decay when otherwise inhibitory
secondary structures are present. For example, increased polyadenylation due to
overexpression of pcnB in E. coli destabilizes rpoS, trxA, lpp, ompA, and total RNA.
Although polyadenylation promotes bacterial mRNA decay, the presence of these

14
 PROKARYOTIC MESSENGER RNA

elements may also recruit poly(A)-binding proteins, such as CspE, which when bound to
poly(A) tails interfere with RNase activity. Nonetheless, RNase E can remove poly(A)
tails by endoribonucleolytic cleavage which may eliminate inhibitory poly(A) binding
proteins. As mentioned above, RNase II degrades polyadenylated mRNAs; however,
polyadenylated rpoS is stabilized by RNase II. In vivo, RNase II shortens the rpoS
poly(A) tail making the transcript less susceptible to PNPase-mediated
decay.Collectively, ribonucleases, RNA binding proteins, and noncoding RNA molecules
dynamically regulate E. coli gene expression by affecting mRNA stability. As will
become evident (discussed below), these factors likely modulate mRNA stability in the
model gram-positive organism B.[7]

9.Difference Between Prokaryotes and Eukaryotes mRNA:-

Messenger RNAs of prokaryote and eukaryote cells have several distinguishing

features.

(1) The mRNAs of many bacteria and bacteriophages are polygenic or polycistronic. A
polycistronic mRNA is transcribed by the several structural genes of an operon. It
contains several sites for initiating and terminating polypeptide synthesis. On the other
hand all known eukaryotes have only one site for initiation of protein synthesis. Thus
eukaryote mRNAs are monocistronic.
(2) In most bacterial mRNAs translation begins while the mRNA is still being transcribed
on DNA. In eukaryotes the mRNA transcribed on the chromosomes passes through the
nuclear pores into the cytoplasm. Here it forms complexes with ribosomes, which
synthesize proteins. Thus translation usually begins only after transcription is completed.

(3) Prokaryote mRNA is very short lived. It is constantly under going breakdown to its
constituent ribonucleotides by ribonucleases. In E. coli the average half life of some
mRNAs is about two minutes. In bacteria mRNA may be so short lived that while one
end is translating proteins the other end may be undergoing breakdown.
The short life of bacterial mRNAs has been explained on the grounds that it provides
greater flexibility to the bacteria by adjusting to changing environmental conditions. The
short life of its mRNAs enables a bacterium to synthesize different enzymes in response
to environmental changes. In general, eukaryote mRNAs have longer half lives than
bacterial mRANAs. In other words eukaryote mRNAs are metabolically
stable.Mammalian reticulocytes which have lost their nuclei synthesize haemoglobin for
hours or even days by utilizing mRNAs which were transcribed when nuclei were present
mRNA stability enables eukaryote cells to have a more permanent protein complement.
This permits differentiation of cells to occur.
(4) In prokaryotes the mRNAs undergo very little processing after being transcribed.
There is a very short time interval between transcription and translation. In fact
considerable translation may already take place before completion of transcription, and
degradation of mRNA may begin. In eukaryotes the transcribed mRNA undergoes
considerable processing before maturem RNA is formed.

Processing consists of:

15
 PROKARYOTIC MESSENGER RNA

(i) polyadenylation or addition of 180-200 adenylate residues at the 3' end forming a long
poly(A)chain,

(ii) capping or formation of a 'cap' at the 5' end by condensation of a guanylate residue,
and (iii) methylation or the addition of methyl groups to some nucleotides. Transcribed
precursor RNA, called heterogenous nuclear RNA (hnRNA), may be 5,000 to 50,000
nucleotides long.
After cleavage and degradation the mature mRNA formed may be just a fraction of the
original hnRNA length. Thus in duck reticulocytes the precursor hnRNA is X 10 to X
100longerthanmaturehaemoglobinmRNA.

(5) With the exception of histone mRNAs, most mRNAs of eukaryotic cells have the 3'
terminal poly (A) chain of 180-200 nucleotides referred to above. This poly (A) chain
shortens with age and probably functions as an mRNA stabilizer. Prokaryotic mRNAs do
nothaveapoly(A)tailatthe3'end.

(6) Eukaryote mRNAs which are capable of being translated begin with the sequence
m7G(5')ppp(5')N, where m7G is a methylguanosine residue and (5')ppp(5') a 5'-5'
triphosphate linked to a base at the 5' end.[3]
. subtilis and the human pathogen S. aureus as well.[6]

16
 PROKARYOTIC MESSENGER RNA

References:
[1] http://en.wikipedia.org/wiki/Messenger_RNA
[2] http://www.answers.com/topic/rna
[3http://www.microbiologyprocedure.com/ribose-nucleic-acid/structure of-
mRNA.htm
.[4] www.ehow.com/tag/mrna/
[5] http://en.wikipedia.org/wiki/Prokaryotic_translation
[6] http://www.ncbi.nlm.nih.gov/pmc/articles/PMC432886/
[7] http://www.hindawi.com/journals/ijmb/2009/525491.html

17