Box 9.1 (continued)
are based on homology with known ncRNA. These
prediction tools cannot detect antisense ncRNAs
encoded by ncRNAs genes and proteins that are
not conserved, which genomes are not yet
sequenced. However, hundreds of ncRNAs have
been predicted and are waiting to be validated
experimentally (Altuvia 2007).
• Direct sequencing of ncRNAs: the RNAs are
separated by electrophoresis after metabolic
labeling or end-labeling.
• Microarray-based genome oligonucleotide. Several
experimental conditions have to be tested for
targeting ncRNAs that are expressed in response
to certain stimuli.
• RNomics: RNAs of a size between 20 and 500
bases are retro-transcribed, isolated by denaturing
PAGE, and cloned. Reverse transcription is still not
effective on highly structured RNA or modified;
thus, only transcribed and abundant RNAs will be
identified (Vogel et al. 2003).
• Genomic SELEX (“systematic evolution of ligands
by exponential enrichment”): This is an experimental approach for identifying protein-binding
ncRNAs, independently of the conditions of their
expression. The ncRNA-protein complex may be
essential for the regulatory function of ncRNAs that
act as antisense regulators that interact with the
protein Hfq (Wassarman et al. 2001). Enrichment
steps are based on a ncRNAs size selection and
affinity for protein ligands (Lorenz et al. 2006).
Functional Approach of RNomics
The function of ncRNAs identified by different
techniques mentioned above could be elucidated by
different approaches (Hu ¨ttenhofer and Vogel 2006):
since most functional ncRNAs are part of ribonucleoprotein complex (RNP), the protein component of
ncRNAs can be sought and identified by affinity
trapping technique by labeling the ncRNA. Some
ncRNAs act as antisense mRNA; the search for complementary ncRNAs by bioinformatics tools should
identify the targets of these ncRNAs. Overexpression
or construction of mutants that do not express the
ncRNA is expected to highlight the phenotypes
associated with them.
Mode of Action of ncRNA
The ncRNAs interact with other RNA molecules either
to stabilize them and translate them or to destabilize
them and lead to their degradation (Fig. 9.15).
NcRNA-mRNA interactions are reinforced by Hfq-like
(continued)
Box 9.1 (continued)
proteins. The ncRNA can also interact directly with
proteins to sequester them (Box Fig. 9.1).
Base pairing with the transcript (RNA) target:
• The matching is perfect if the ncRNAs are encoded
(in cis) by the complementary strand of the target
RNA. This mainly concerns the plasmid ncRNAs
that act as antitoxins to toxins: it is the case of the
hok/sok (“host killing/suppressor of killer”) locus –
the ncRNA sok and the hok mRNA form a duplex
that is rapidly degraded by RNase III (Gerdes et al.
1992). If the bacterium loses the plasmid, the level
of sok decreases faster than that of hok which is
translated into the Hok protein, leading to cell death
(Hayes 2003).
• Pairing is imperfect if the target RNA and ncRNAs
are encoded (in trans) by genes located in different
parts of chromosome as in the case of ncRNA
RyhB or SgrS which respectively control the level
of iron and glucose-phosphate in the cells of E. coli.
A ncRNA can target multiple mRNAs; this is the
case for RyhB, which regulates the level of ironcontaining proteins expression in cases of iron
deficiency (Masse ´ and Gottesman 2002).
Modification of the Activity of Proteins
6S RNA from E. coli is one of the first ncRNA
detected and identified (Lee et al. 1978). Its expression
level increases when the medium is depleted
and bacteria enter the stationary phase. 6S RNA
binds to RNA polymerase (RNA polymerase-σ70) by
mimicking the DNA, thus preventing gene transcription that is dependent upon the 70s factor (Lee et al.
1978). 6S RNA is essential for the transition from
exponential phase to stationary phase and for
long-term survival of bacteria.
Modulation of Translation
Most ncRNAs modulate the stability of transcripts
and regulate translation by masking the ribosome
binding site on the mRNA. Some ncRNAs form a
ribonucleoprotein complex with the protein Hfq and
RNaseE to inhibit translation of target mRNA and to
accelerate its degradation (Morita et al. 2005). This is
the mode of action of RhyB and SgrS; a family of
homologous proteins to CsrA (“carbon storage regulator A”) in E. coli and to RsmA (“repressor of
stationary phase of metabolites”) in species of Pseudomonas is involved in the regulation of carbon
metabolism, virulence genes, mobility, and many
other functions. These proteins bind to the 5
0 end of
(continued)
9 Adaptations of Prokaryotes to Their Biotopes and to Physicochemical Conditions. . .
295
are based on homology with known ncRNA. These
prediction tools cannot detect antisense ncRNAs
encoded by ncRNAs genes and proteins that are
not conserved, which genomes are not yet
sequenced. However, hundreds of ncRNAs have
been predicted and are waiting to be validated
experimentally (Altuvia 2007).
• Direct sequencing of ncRNAs: the RNAs are
separated by electrophoresis after metabolic
labeling or end-labeling.
• Microarray-based genome oligonucleotide. Several
experimental conditions have to be tested for
targeting ncRNAs that are expressed in response
to certain stimuli.
• RNomics: RNAs of a size between 20 and 500
bases are retro-transcribed, isolated by denaturing
PAGE, and cloned. Reverse transcription is still not
effective on highly structured RNA or modified;
thus, only transcribed and abundant RNAs will be
identified (Vogel et al. 2003).
• Genomic SELEX (“systematic evolution of ligands
by exponential enrichment”): This is an experimental approach for identifying protein-binding
ncRNAs, independently of the conditions of their
expression. The ncRNA-protein complex may be
essential for the regulatory function of ncRNAs that
act as antisense regulators that interact with the
protein Hfq (Wassarman et al. 2001). Enrichment
steps are based on a ncRNAs size selection and
affinity for protein ligands (Lorenz et al. 2006).
Functional Approach of RNomics
The function of ncRNAs identified by different
techniques mentioned above could be elucidated by
different approaches (Hu ¨ttenhofer and Vogel 2006):
since most functional ncRNAs are part of ribonucleoprotein complex (RNP), the protein component of
ncRNAs can be sought and identified by affinity
trapping technique by labeling the ncRNA. Some
ncRNAs act as antisense mRNA; the search for complementary ncRNAs by bioinformatics tools should
identify the targets of these ncRNAs. Overexpression
or construction of mutants that do not express the
ncRNA is expected to highlight the phenotypes
associated with them.
Mode of Action of ncRNA
The ncRNAs interact with other RNA molecules either
to stabilize them and translate them or to destabilize
them and lead to their degradation (Fig. 9.15).
NcRNA-mRNA interactions are reinforced by Hfq-like
(continued)
Box 9.1 (continued)
proteins. The ncRNA can also interact directly with
proteins to sequester them (Box Fig. 9.1).
Base pairing with the transcript (RNA) target:
• The matching is perfect if the ncRNAs are encoded
(in cis) by the complementary strand of the target
RNA. This mainly concerns the plasmid ncRNAs
that act as antitoxins to toxins: it is the case of the
hok/sok (“host killing/suppressor of killer”) locus –
the ncRNA sok and the hok mRNA form a duplex
that is rapidly degraded by RNase III (Gerdes et al.
1992). If the bacterium loses the plasmid, the level
of sok decreases faster than that of hok which is
translated into the Hok protein, leading to cell death
(Hayes 2003).
• Pairing is imperfect if the target RNA and ncRNAs
are encoded (in trans) by genes located in different
parts of chromosome as in the case of ncRNA
RyhB or SgrS which respectively control the level
of iron and glucose-phosphate in the cells of E. coli.
A ncRNA can target multiple mRNAs; this is the
case for RyhB, which regulates the level of ironcontaining proteins expression in cases of iron
deficiency (Masse ´ and Gottesman 2002).
Modification of the Activity of Proteins
6S RNA from E. coli is one of the first ncRNA
detected and identified (Lee et al. 1978). Its expression
level increases when the medium is depleted
and bacteria enter the stationary phase. 6S RNA
binds to RNA polymerase (RNA polymerase-σ70) by
mimicking the DNA, thus preventing gene transcription that is dependent upon the 70s factor (Lee et al.
1978). 6S RNA is essential for the transition from
exponential phase to stationary phase and for
long-term survival of bacteria.
Modulation of Translation
Most ncRNAs modulate the stability of transcripts
and regulate translation by masking the ribosome
binding site on the mRNA. Some ncRNAs form a
ribonucleoprotein complex with the protein Hfq and
RNaseE to inhibit translation of target mRNA and to
accelerate its degradation (Morita et al. 2005). This is
the mode of action of RhyB and SgrS; a family of
homologous proteins to CsrA (“carbon storage regulator A”) in E. coli and to RsmA (“repressor of
stationary phase of metabolites”) in species of Pseudomonas is involved in the regulation of carbon
metabolism, virulence genes, mobility, and many
other functions. These proteins bind to the 5
0 end of
(continued)
9 Adaptations of Prokaryotes to Their Biotopes and to Physicochemical Conditions. . .
295