Title: RNA Switches Genetic Research Tools
1RNA Switches Genetic Research Tools
RAVINDER NAGPAL1, MALVIKA MALIK1, MONICA PUNIYA2,
AARTI BHARDWAJ3, SHALINI JAIN4 and HARIOM
YADAV4 1Dairy Microbiology, 2Dairy Cattle
Nutrition, 4Animal Biochemistry, National Dairy
Research Institute, Karnal 132001, Haryana, 3
Meerut Institute of Engineering and Technology,
Meerut-250002, U.P., India. Email
yadavhariom_at_gmail.com
2Role of RNA inside the Cell
- According to traditional central dogma of
genetics RNA is the intermediate carrier of
genetic information between DNA and Protein. - DNA RNA
PROTEIN
Transcription
Translation
3New roles discovered for RNA
- Certain RNA molecules, ribozymes, could catalyze
biochemical reactions, a job previously thought
to be the exclusive province of enzymes, which
are proteins. (Kruger et al, 1980) - Certain RNA molecules could behave like another
type of protein antibodies. Researchers
synthesized RNA molecules - now known as
aptamers- that, like antibodies, can latch on
tightly to specific target molecules.
- (Ellington and Szostak, 1990)
4contd...
- Certain RNA sequences can directly sense
environmental factors and small molecule
metabolites, this allow the associated mRNA to
regulate their own transcription or translation.
(Breaker, 2002) - These self regulatory messages are called as RNA
sensors and RNA switches.
5RNA Switches
- RNA switches are mRNAs that sense the environment
directly, shutting themselves down in response to
particular chemical clues. - Breaker, Nudler, Yura and Cossart laboratories
report that specific RNA sequences can act as
environmental sensors of vitamin cofactors
(including vitamins B1, B2 and B12) and
temperature, which allow them to directly
regulate the transcription or translation of
associated mRNAs.
6contd...
- Distinct RNA molecules directly perform or
mediate enzymatic processes such as RNA cleavage,
splicing and translation. - Non-coding RNAs are involved in a tremendous
variety of gene regulatory mechanisms that
operate at both the DNA and mRNA level . - Seven types of RNA switches have been found in
bacteria so far. These include switches
controlling the manufacture of the vitamins B1
and B2 and the nucleotide guanine.
7Mode of action of RNA Switches
- RNA Switches can regulate gene expression at both
transcription as well as translation level. - At transcription level they can induce premature
termination of mRNA transcript. - At translation level the initiation of protein
synthesis is stopped by these switches. - In certain cases RNA switches have self-cleavage
property, thus regulating their own expression. - RNA switches can also act as thermo sensors,
regulating gene expression according to the
temperature change.
8Anti-term.
Term.
Anti-Anti-Term.
RNA poly.
DNA
LIGAND
RNA poly.
Regulation by Transcriptional Termination
9Examples of Transcription Termination Mechanism
- Transcription termination mechanism has been
reported functional in the regulation of rib and
thi operon in Bacillus subtilis.
(Mironova et al, 2002) - rib operon - riboflavin biosynthesis.
- thi operon - thiamine biosynthesis.
- In case of rib operon the 5 Untranslated Region
of the mRNA transcript can fold into two
confirmations, depending upon the presence or
absence of FMN.
10contd...
- In low concentration/absence of FMN the nascent
transcript come out of RNA polymerase and attain
a confirmation that allow the transcription to be
completed. - In presence of FMN, it binds to the nascent
transcript and transcript folds in such a way
that the transcription is terminated , resulting
in premature transcript which leads to no gene
expression.
11Anti-anti-SD
Anti-SD
SD
AUG
Ribosome
LIGAND
Anti-SD
SD
Anti-anti-SD
AUG
Regulation by Translational Initiation Inhibition
12Example of Translation Inhibition Mechanism
- Translation initiation inhibition mechanism has
been proposed for regulation of thiM and thiC
genes required for biosynthesis of thiamin.
(Mandal and Breaker, 2004) - The translation initiation for these genes is
sensitive to the presence of thiamin. - Binding of thiamin to the mRNA precludes the
Ribosome Binding Site from ribosome subunit
access.
13Observations
- Gram-positive bacteria are more likely to couple
these elements to a terminator/anti-terminator
system, that is at the level of transcription. - Gram-negative bacteria more typically link these
elements to a SD-sequestration mechanism, that is
at the level of translation.
14contd...
- This type of regulation is found to be economic
for the bacterial system as- - In Gram positive bacteria the genes for one
biosynthetic pathway are arranged in a cluster,
therefore they are regulated at the transcription
level. - In Gram negative bacteria the genes for one
biosynthetic pathway are scattered along the
whole genome and are therefore regulated at the
translation level.
15Self-cleavage of the RNA molecules
- The regulation of glmS gene(glutamine-fructose-6-p
hosphate amidotransferase)in B. subtilis is by
associated activity of ribozyme and riboswitches. - In this case the 5 UTR of glmS binds with
gluosamin-6-phosphate, product of GlmS activity
and start acting as ribozyme, due to
conformational change. - This results in the self cleavage of the mRNA by
an internal phosphoester transfer.
16Secondary Structure of self cleavage RNA molecule
17Activation of gene expression by RNA Thermo
sensors
- There are two well-studied examples of
temperature-dependent regulation of gene
expression and/or activity- - during the heat shock response
- during pathogenic invasion
- Almost all the known thermo sensors regulate the
gene expression at the translational level.
18Translation regulation by an mRNA Thermo sensor
Ribosome
mRNA
SD
AUG
Temperature Increase
SD
AUG
19Examples of RNA Thermo sensors
- Translation of LcrF, a general activator of
virulence-related gene expression in Yersinia
pestis, was found to be thermally regulated.
(Hoe and Goguen, 1993) - A new report from the Cossart lab (2002) now
provides strong evidence for an RNA thermo sensor
that regulates translation of PrfA, a general
activator of virulence genes in a pathogenic
variety of Listeria, L. monocytogenes.
20Regulation of virulence gene in L. monocytogenes
- In L. monocytogenes prf A gene is responsible for
the virulence of the organism. - The prfA gene is transcribed at both 30C and
37C. - But it is translated only at 37C.
- sequences in the 5' UTR of the prfA mRNA could
form an extended hairpin that includes the SD
sequence. - At 37C, when the pathogen enters into the host
system, this structure is destabilized the SD
sequence is free initiation of translation.
21STRUCTURE OF RNA SWITCHES AND THEIR LIGANDS
22 Natural small molecule-regulated RNA switches
                                              Â
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23Advantages of RNA switches for the organisms
possessing them
- Stringent control RNA switches are very specific
to their substrate. eg. recognition of TPP by THI
boxes is 1000 times greater than for either
thiamine or thiamine monophosphate.
( Winkler et. al. 2004) - Ligand can be reused Binding of the substrate to
the RNA molecule is required for a short time,
after this the ligand can be reused for the same
or different purpose. - Only one gene is involved There is no
involvement of another gene product, which
reduces the effect of mutation that will
deregulate the mechanism.
24 Gene expression regulation by RNA in Eukaryotes
- De-repressor RNA
- Recent data indicate that small non-coding RNA
species function as co-activators of eukaryotic
gene transcription . - Small activator or de-repressor RNA molecules
play a role in initiating and stabilizing
transcription bubbles, mRNA synthesis by RNA
polymerase. (Frenster, 1965)
25dsRNA as a regulator of gene expression
                                              Â
            Â
- ds RNA has role in several chromatin and/ or
genomic DNA modifications, which lead in the
regulation of specific genes. - ds RNA dependent mechanism can act at both
transcriptional as well as post transcriptional
levels. - This type of gene expression is given different
names in different organisms. - RNA interference (RNAi) , in case of animals.
- Post transcriptional gene silencing (PTGS) , in
case of plants. - Quelling, in case of filamentous fungi.
26Other RNAs involved in regulation of gene
expression
- micro RNA (mi RNA)- a class of noncoding small
RNA identified for their role in translational
repression in some animals. - short hairpin RNA (sh RNA)- a special class of
RNA that becomes double stranded by folding of
the RNA strand over itself, has been shown to be
responsible for the transcriptional regulation.
27Applications of RNA switches in genetic research
- RNA switches can be used to create small
molecule-regulated transgenes, which may allow
researchers to manipulate expression of any
individual construct within a battery of
simultaneously introduced experimental
constructs.
(Werstuck and Green,
1998) - We can imagine exploiting RNA thermoregulation to
create heat-inducible transgenes.
(Brand and Perrimon,
1993)
28contd...
- Aptamers have been placed within 5' UTRs and
shown to inhibit gene expression upon
introduction of the appropriate ligand.
(Werstuck and Green, 1998) - Self-cleaving RNAs can be placed under allosteric
control by various small molecules, which can
then be used to analyze the composition of
chemical and biological mixtures .
(Seetharaman et al, 2001) - RNA switches can be used in gene therapy,
allowing the patient to take pills to switch
introduced gene on or off.
29contd...
- It may be possible to design drugs using RNA
switches cognate ligands that would
constitutively repress associated gene activity. - Such compounds would efficiently inhibit
bacterial growth by simultaneously repressing
multiple components in a given biosynthetic/metabo
lic/transport pathway. - Such compounds might be likely to have relatively
low toxicity, as they would be designed to target
RNA, not protein.
30RNA on a Chip
- In 1995, Breaker successfully engineered
RNA-based molecular switches. - A molecular switch is a molecule that turns on or
off by another molecule or compound. - With dozens of these switches on hand, Breaker
created an array of biosensors that use RNA to
measure or detect compounds.
31RNA Biochips An array of RNA molecular switches
constructed using seven distinct
effector-modulated ribozymes based on the
hammerhead self-cleaving RNA.                    Â
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32Array for RNA
- Breaker placed the RNA switches on a gold-coated
silicon surface and arranged them in clusters. - Each switch was designed to bind only to a
specific molecule its target and then to
release a signal that identifies the target
molecule. (In the prototype, the switches
released a radioactive signal.) - This array of RNA switches was tested on a
variety of complex mixtures. In one experiment
different strains of E. coli found in bacterial
cultures were successfully identified.
33- The array's ability to simultaneously identify a
potentially large number of compounds, combined
with the precise exclusivity of each switch, adds
up to a recipe for a powerful and wide-ranging
laboratory on a dime-sized slice of silicon.
34RNA Super chip
- Future RNA chips, capable of revealing the
molecular composition of complex mixtures-like
blood serum and industrial waste-far more
comprehensively than current biochips can be
synthesized. - Advanced versions of RNA biochip could be used
for many different targets like drugs, toxins and
metabolites, as well as proteins and nucleic
acids.
35Benefit of using RNA biochip
- Benefit of RNA switches is their ability to
withstand the sometimes unpredictable and harsh
environment outside the lab as compared to a
protein biochip. - Breaker's RNA switches have been engineered to
refold back to their original form after heating,
this snap-back character will give RNA biochips a
considerable advantage for use in more exotic
test environments.
36Obstacles
- Manufacturing costs .
- The chemical stability of the switches .RNA is
vulnerable to certain chemicals often found in
test situations that can disintegrate a switch. - The finer points of molecular recognition .
- This technology is so new that it is unclear just
how many different compounds it will prove
possible to recognize .
37Future Prospects
- RNA switches must be engineered to release a
fluorescent, rather than a radioactive signal. - Use of an RNA chip in diverse fields like
chemical engineering, environmental science, and
even biological and chemical warfare defense. - Use of RNA chip in the detection and
identification of pathogens.
38(No Transcript)
39Conclusion
- Apart from being the carrier of genetic
information , RNA can also act as the regulator
of expression this information. These regulators
are called as RNA switches. - RNA switches have been discovered to be involved
in the regulation of gene expression in
prokaryotes. - RNA switches can be used in genetic research to
study the effect of environmental changes on
particular gene expression.
40- RNA switches can be used to in gene therapy and
as drugs. - Researchers are trying to synthesize artificial
RNA switches and we can hope that in future we
will be able to use RNA switches not only in
genetic research but also in antimicrobial
therapy, as biosensors for different chemicals
and in detection of pathogenic microorganisms.