This presentation is about Riboswitches and Riboswitches mediated regulation. Riboswitches are the small mRNA element that has tertiary structure and regulate the down stream genes in the same mRNA by interacting with small metabolites and metal ions.Various types of regulatory mechanism and structure and ligand binding of some important riboswitches are given here.Like TPP,PURINE AND FMN riboswitches. Also the role of some tandem and cooperative riboswitches are given here. Applications of Riboswitches are also given here like drug targets. Some future challenges are also given here.

1. Swastik Kar-16MS192
3rd Year BS-MS
IISER KOLKATA

2.  Introduction
 History and Discovery
 Structure of Riboswitches
 Types and Subfamilies
 General Mechanism
 Structure and Function of some important
Riboswitches.
 Regulation by RNA switches.
 How Riboswitches can affect human cells
 Future Challenges and Perspectives.
 References

3.  What is a riboswitch: Genetic regulation by RNA
is widespread in bacteria. One common form of
riboregulation in bacteria is the use of ribonucleic
acid sequences encoded within mRNA that
directly affect the expression of genes encoded in
the full transcript (called cis-acting elements
because they act on the same molecule they're
coded in). These regulatory elements are known as
riboswitches and are defined as mRNA elements
that bind metabolites or metal ions as ligands and
regulate mRNA expression by forming alternative
structures in response to this ligand binding.

4. History and Discovery
Riboswitch was first
named by Dr. Ronald
Breaker in 2002.
Until 2002, Dr. Breaker
first demonstrated that
mRNAs can bind
metabolites directly in
the absence of proteins
and he also developed a
useful method, in-line
probing to detect the
conformational change
of mRNA.

6. 1) Ligand binding domain.
2) Highly conserved in sequence and structure even
among widely diverse organism and likewise
conserved when multiple variants of a given
riboswitch are present in a single organism.
3) Upon binding of the target metabolite to this
aptamer conformational changes results that
modulate expression of downstream genes carried
by the mRNA.
4) The aptamer domains are in range from ~70 to
~200 nucleotides.

7. 1) Less conserved in sequence ,structure and
size.
2) Shine-dalgarno sequence locates in this
domain.

8. Riboswitches
architecture
Schematic representations
of a “straight” junctional
fold (A), observed in type
Ia riboswitches; an
“inverse” junctional fold
(B) from type Ib
riboswitches; and a
pseudoknot fold (C), which
is characteristic for type II
riboswitches. Hot pink and
blue shading depict ligand
binding sites and long-
distance tertiary
interactions, respectively.
The magenta segment in
helix P1 designates regions
capable of alternative base
pairing.

9. Ligand name Riboswitch
family/class
Function
S-
adenosylmethionine
SAM riboswitch Methionine biosynthesis, cysteine biosynthesis,
methylene tetrahydrofolate
reductase, SAM synthesis
Thiamine
pyrophosphate
TPP riboswitch Thiamine synthesis and phosphorylation,
and transport
Guanine Purine/G riboswitch Purine synthesis and transport
Adenine Purine/A riboswitch Purine synthesis and transport
2'-Deoxyguanosine Purine/dG riboswitch Purine synthesis and transport
Lysine Lysine Riboswitch Lysine synthesis and transport, lysine
catabolism
Vitamin B12 Vitamin B12
riboswitch
Cobalamin synthesis and transport,
cobalt transport, aerobic and anaerobic
ribonucleotide reductase
FMN FMN riboswitch Riboflavin biosynthesis and transport
Glycine Glycine riboswitch Glycine catabolism and efflux
GlcN6P riboswitch GlcN6P riboswitch GlcN6P synthesis

10. General Mechanism
(In Prokaryotes)
Left: metabolite binding most
often prevents formation of the
antiterminator hairpin
(complementary RNA regions in
light blue) and promotes
formation of the alternative Rho-
independent termination hairpin
(middle) or Rho binding site
(bottom) that causes premature
transcription termination. Center:
in some cases, bound metabolites
stabilize the antiterminator
hairpin that allows RNA
polymerase (Pol) to complete
transcription of the gene (bottom).
Right: expression of open reading
frames (ORF) can be repressed by
sequestration of the ribosome
entry site (RBS or Shine-Dalgarno
sequence, SD, dark blue) and
blockage of translation initiation
(middle). Metabolite binding to
some riboswitches facilitates
formation of the SD antisequester
hairpin that opens up SD for
ribosome binding and translation

11. Degradation of mRNA after ribozyme cleavage
In gram-positive
bacteria, bound
GlcN6P induces
cleavage by
the glmS riboswitch-
ribozyme in the 5′
UTR. The 5′-OH of
the resulting
fragment stimulates
degradation of the
message by RNase J.

12. Alternative self-splicing
C. difficile exploits an allosteric
ribozyme-riboswitch that
combines self-splicing and
translation activation. Left: in
the absence of c-di-GMP, the
intron uses GTP cleavage site 2
(black arrow, GTP2), thus
yielding RNAs with truncated
SDs that are not expressed.
Right: binding of c-di-GMP to
the riboswitch in the presence
of GTP promotes cleavage at
site 1 (green arrow, GTP1) and
self-excision (green arrowed
lines) of the group I self-
splicing intron using splicing
sites shown in green circles.
This self-cleavage brings
together two halves of the SD,
and the resulting mRNA is
efficiently translated.

13. Cis-Transcription termination and trans-translation
repression
In L. monocytogenes, the
SreA and SreB riboswitches
form antiterminator
hairpins and allow normal
transcription in the absence
of SAM. Binding of SAM
causes transcription
termination. The resulting
mRNA fragments base pair
with the 5′ UTR of the
mRNA and functions
in trans as an antisense
sRNA that destabilizes the
target transcript, thus
reducing protein
production.

14. TPP-Dependent Riboswitches Regulation in Eukaryotes
In fungi, complementary base
pairing results in preferential use
of the distal set of splicing sites
(black open circles) and
elimination of the region
between black dashed arrowed
lines (left). The resulting mRNA
is translated to yield full-length
product. TPP binding to the
riboswitch stabilizes the
riboswitch fold, precludes the
complementary base pairing, and
opens different set(s) of splicing
sites (green circles) that are
otherwise sequestered. Splicing
at the alternative splice sites
removes sequences between the
green arrowed lines. The
resulting mRNAs retain micro
ORFs, which preclude translation
of the main ORF located
downstream of the micro ORF
(right).

15. TPP based Alternative Splicing and translation
termination
In algae, mRNA splicing
in the absence of TPP
eliminates a stop codon
located within a
riboswitch sequence.
TPP binding promotes
alternative splicing
events that introduce a
premature termination
codon and disrupts
translation of the ORF.

16. TPP based splicing and mRNA degradation in higher
plants
In higher plants,
sequestration of splice sites
in the absence of TPP causes
retention of the mRNA
processing site
(polyadenylation signal,
yellow rhomb) and yields
stable mRNA with a short 3′
UTR. TPP binding to the
riboswitch sensor exposes
the 5′ splice site, causing the
removal of the fragment
between the green arrows
containing the
polydenylation signal. The
resulting mRNA contains
long and less stable 3′ UTR,
which compromises protein
production.

17. Purine riboswitches: Global Structure
The purine riboswitch family
includes the adenine, guanine,
and 2'-deoxyguanosine
classes..The global architecture of
the RNA in a purine riboswitch
is defined by the organization of
the three conserved helices that
make up the secondary structure.
Two of the RNA helices form a
coaxial stack, meaning that one
helix sits on top of the other, and
they are collinear. This pairing is
the basis for their names, P1 and
P3. The third helix (P2) is
adjacent to P3, and the terminal
loops of P2 and P3 together form
a tertiary structure called a loop-
loop. A complex set of
interacting helices and loop
formations defines the overall
three-dimensional fold of the
purine riboswitch aptamer
domain where ligands bind.

18. Purine riboswitches: Ligand Binding in the Aptamer
Domain
The three-way helical junction
where P1, P2, and P3 meet is the
ligand-binding pocket of a
purine riboswitch. This region of
the RNA is defined by a series of
noncanonical base interactions.
For example, in the P1 helix
proximal to the ligand-binding
site, a base triple interaction is
observed in most purine
riboswitches .This triple, as well
as most other unusual base
triples, is typically composed of a
Watson-Crick pair (A21-U75)
interacting with a third base
(C50). At the center of the
junction, a pyrimidine (Y) at
position 74 forms a Watson-Crick
pairing interaction with the
ligand, which is further
surrounded by other conserved
residues. The identity of this
pyrimidine residue (cytosine or
uridine) is the basis for
specificity between the guanine
and adenine classes.

19. Purine riboswitches: Regulatory mechanism
The guanine-specific riboswitch
from the xpt-pbuX operon of B.
subtilis. Guanine (G) binding
involves base pairing to a
cytidine (C) residue of the
aptamer and causes formation
of an intrinsic terminator stem
that turns off gene expression.
When G is absent, the anti-
terminator stem is formed at the
expense of P1. The adenine-
specific riboswitch from the
ydhL gene of B. subtilis74. A
uridine (U) residue is responsible
for base pairing to the ligand.
Adenine (A) binding prevents
formation of the terminator,
thereby causing gene expression
to switch on. In this case, the
antiterminator structure is part
of the aptamer (stems P1 and
P2). In the absence of adenine,
some sequences of P1 and P2
now form a part of the intrinsic

20. FMN Riboswitch: Structure and Lingand binding

22. Binding kinetics for simple riboswitches, or complex riboswitches that use cooperative binding or a tandem
architecture using riboswitches of from the same class. (A) Dose-response curve for a typical riboswitch carrying a
single aptamer that functions perfectly. Note that the plot represents the performance of a population of individual
riboswitch molecules where [R] represents the fraction of riboswitches causing gene expression change on ligand
binding. Ligand concentration [L] is in arbitrary units, and T50 represents the concentration of ligand needed to half-
maximally modulate gene expression. (B) Comparison of the dose-response curve for a simple riboswitch (one aptamer
and one expression platform) versus a cooperative riboswitch (two aptamers and one expression platform). The curve
for the cooperative riboswitch reflects perfect cooperativity between the aptamers and a Hill coefficient (n) of two.
Note that [Y], the fraction of riboswitches bound by ligand, is equivalent to [R] if ligand binding always triggers a
change in gene expression. (C) Comparison of the dose-response curve for a simple riboswitch versus a tandem
arrangement of independently functioning riboswitches of the same class and
near identical T50 values. Other annotations are as described in A and B.

23. Cooperative glycine riboswitch system that yields a more digital genetic
response in numerous Gram positive bacteria including in the 5′ UTR of
the B. subtilis gcvT gene.

24. Tandem TPP riboswitches from the 5′ UTRof the thiamin metabolism
gene tenA from Bacillus anthracis.

25. Tandem SAM-II and SAM-Vriboswitches identified in ocean bacteria such as
“Cand. P. ubique”.

26. A two-input Boolean NOR logic gate composed of tandem riboswitches for
SAM and AdoCbl located in the metE gene from Bacillus clausii.

27. A Protein-directed RNA Switch in Higher Eukaryotes
Secondary structure of VEGF
HSR predicted by Mfold
shows GAIT element (green),
hnRNP L binding site (red),
and stem stability sequence
(blue). TP is lowest free energy
conformer predicted by Mfold
(left). TS conformer was
generated by introducing
experimentally-determined
base-pairing constraints in
GAIT element stem (right).
Strong and weak RNase
cleavage sites are marked by
red and blue circles,
respectively. Key signature
cleavage sites are indicated (*,
**)

29. Since these fascinating riboswitches are mechanisms
specific to bacteria, it may be difficult determine how
relevant they are to humans and human health. However,
their role in regulating transcription in bacteria makes
them enticing targets for the development of novel
antibiotics aimed at stopping bacterial pathogens from
flourishing inside the people they infect. Because
riboswitches control genes essential for bacterial survival,
or genes that control the ability of bacteria to succeed at
infection, a drug designed to affect a riboswitch could be a
powerful tool for shutting down pathogenic bacteria. In
fact, many antimicrobial compounds affect RNA directly,
and many commonly used antibiotics inhibit translation
by targeting bacterial ribosomes through binding
interactions with ribosomal RNA . In addition, some
compounds bind to the lysine, TPP, and FMN riboswitch
classes and slow bacterial cell growth

30.  Bioinformatic searches have identified many
conserved mRNA elements that could potentially
function as riboswitches but were missing their
validated ligands. Some of these so-called
‘‘orphan’’ riboswitches are widespread in nature
and may be associated with sensing of novel
chemical cues.
 Another hurdle in riboswitch validation is that the
relationship between the structures of riboswitches
and the nature of their cognate metabolites are not
well understood, and growing evidence suggests
that such interconnection may not exist.

31.  Arnaud, M. et al. In vitro reconstitution of transcriptional
antitermination by the SacT and SacY proteins of Bacillus
subtilis. Journal of Biological Chemistry 271, 18966–18972 (1996)
 Aymerich, S. & Steinmetz, M. Specificity determinants and structural
features in the RNA target of the bacterial antiterminator proteins of
the BglG/SacY family. PNAS 89, 10410–10414 (1992)
 Babitzke, P. & Yanofsky, C. PNAS 90, 133–137 (1993)
 Batey, R. T., Gilbert, S. D. & Montange, R. K. Structure of a natural
guanine-responsive riboswitch complexed with the metabolite
hypoxanthine. Nature432, 411–415 (2004)
 Blount, K. F. & Breaker, R. R. Riboswitches as
antibacterial drug targets. Nature Biotechnology 24, 1558–1564 (2006)
 A decade of Riboswitces.
 A stress-responsive RNA switch regulates VEGF expression