RNA binds ligands OR Ligands bind RNA.
The Riboswitch is functionally separated into the ligand binding APTAMER and the
decision-making EXPRESSION PLATFORM
Purine riboswitch
TPP riboswitch
SAM riboswitch
glmS ribozyme
Part of the proposed mechanism involves co-transcriptional folding of the
riboswitch and coordinated binding of its ligand. A first probing of this
coordination used the FMN riboswitch (Wickiser et al., 2005. Mol Cell 18:49-60.)
How were the ligand interactions demonstrated?
FMN
nt 136
nt 113/114
nt 89/90
nt 56
nt 33/34
In-line probing is used by the Breaker
group to characterize the structures of
riboswitches with and without their ligands.
The principle: spontaneous cleavage of the
phosphodiester backbone in the presence of
Mg+2.
These are internal transesterification reactions that involve the 2’ OH and Mg2+. This reaction
requires a specific geometry (SN2-inline displacement reaction) that does not correspond to
typical conformations in structured regions of an RNA (especially duplex regions).
However, flexible regions of the RNA can access this geometry, at least transiently with
some probability. To enhance the efficiency of the transesterification reaction and thus
backbone cleavage, the reactions contain 20 mM Mg2+ and are run at room temperature for
40 hours.
Why room temperature for 40 hours?
Nucleotides that become less reactive
when FMN is bound.
Binding is specific to FMN.
In vitro termination assays.
Think about RNA folding. There are two aspects of forming a folded RNA
structure. One is the formation of secondary structures. Here, one
secondary structure sequesters the terminator, and one exposes it. Who
wins this contest? Is the winner determined by thermodynamics or
kinetics?
The second aspect is formation of tertiary structures. These can stabilize
a structure, perhaps interact with other RNAs or proteins, or create a
stable ligand binding pocket.
High resolution denaturing gel
around the Shine-Delgarno
sequence +/- FMN.
Proposed mechanism: ii and
iii form without FMN,
exposing the SD element for
the ribosome to bind.
With FMN, the P1 helix is
formed (i and ii) and so iii
pairs with iv.
SAM riboswitch
Whitford et al., 2009. Biophys J 86:L7-L9.
Nucleotide analogue interference mapping (NAIM) uses phosphothioates
that are cleaved by incubation with iodine.
Blue ellipses indicate the site of incorporation of an analog. In Step 3 lane 1 (untreated (UN)) shows
background degradation of the labeled RNA. Lane 2 (unselected (US)) shows the relative incorporation
level for an analog at a specific position (RNA, which did not undergo the selection step, was iodine
cleaved). Lane 3 (unfolded (U)) shows iodine cleavage products for RNA, which derived from the unfolded
population purified from the native gel. Lane 4 (folded (F)) shows iodine cleavage products for RNA, which
was obtained from the folded population eluted from the native gel. (b) Representative preparative native
gels are shown for different analogs. The unfolded and folded populations indicated here were physically
isolated by cutting them out of the gel.
(a) NAIM probing of
magnesiumdependent changes
in the SAM-I
aptamer.
(b) Ligand dependent changes in apparent
melting temperatures mapped on the
secondary structure of SAM-I. Colors
represent the difference in Tm between
bound and free RNAs; positions that
do not display distinct two-state melting
behavior are not included.
(c) NAIM analysis using analogs of
guanine (inosine, yellow) and adenine
(2AP, red; DAP, cyan) identifies specific
nucleotide positions as important for
folding and/or binding.
Adenine RS
Purine riboswitch binding
and folding: a single
molecule force experiment
This riboswitch with its bound ligand has a structure that is more
resistant to disruption. It will be stable in the bound state on a
timescale that allows termination of transcription to occur.
Woodside, Garcia-Garcia & Block (2008) Curr Op Chem Biol 12:640.
The preQ1 (queuosine)
riboswitchRoth et al. (2007)
Nature Struct & Molec Biol 14, 308 317
It is the smallest
riboswitch
currently
known.
Kang et al. (2009) Mol Cell 33: 784
Schematic diagrams of the preQ1 aptamer-domain fold.
Crystal structure of the preQ1 bound form.
Spitale, Torelli, Krucinska, Bandarian, Wedekind (2009) J. Biol.
Chem. 284:11012-11016
PreQ1 riboswitch response. (A) Secondary structure model.
(B) Verification of the equilibrium shift from equally populated, competing hairpins in
the expression platform (fold A: fold B = 50∶50) toward dominant formation of the
downstream hairpin (fold A′: fold B = 20∶80) upon ligand binding by 19F NMR
spectroscopy. Conditions: [RNA] =0.2 mM, [preQ1] = 25 mM, 25 mM sodium
arsenate buffer, H2O⁄D2O = 9⁄1, pH 6.5, 298 K.
Rieder et al., (Micura lab) 2010 PNAS 107(24):10804-9
(A) Schematics of secondary structure rearrangement of
A33Ap labeled variants (34 nt, 61 nt, 70 nt).
(B) (B) Location of A33Ap (red) in the aptamer⁄preQ
(C) (C) Determination of apparent KD value.
Fluorescence changes upon titration of A33Ap 70 nt
labeled variant with preQ1. Normalized Ap
fluorescence intensity plotted as a function of preQ1
concentrations. Changes in fluorescence (F-F0) were
normalized to the maximum fluorescence measured in
the absence of preQ1. The graph shows the best fit to
a single-site binding model. The inset shows
fluorescence emission spectra (λex =308 nm) from
330–480 nm for each preQ1 concentration; RNA = 0.5
µM, 50 mM KMOPS, 100 mM KCl, 2 mM MgCl2, pH
7.0, at 298 K.
(D) Representative fluorescence response of the A33Ap
70 nt variant upon preQ1 addition; preQ1 = 2.0 µM;
mixing was performed with a stopped-flow apparatus.
(E) Plot of observed rate k0 versus ligand concentration
for the three different A33Ap variants as indicated.
Observed rates were determined under pseudofirstorder conditions from three independent stopped-flow
measurements. The slope of the plot yields the rate
constant k.
The Riboswitch Dilemma
RNAP
t1
t2
DNA
t3
What to
do?
t4
terminate
t4
Transcribe/translate
Systematic evolution of ligands by exponential enrichment: RNA ligands to
bacteriophage T4 DNA polymerase.
Tuerk C, Gold L. Science 1990 Aug 3;249(4968):505-10
SELEX: Evolving an aptamer de novo.
RNA and DNA Aptamers are a business opportunity
http://www.aptagen.com/home.aspx
SELEX is an in vitro selection
method to evolve an RNA with
a specific function. It’s been
used to find RNAs that bind
small molecules and proteins,
as well as RNAs with
enzymatic functions (ligase,
polymerase).
We’ll examine a model system for identification of an RNA that binds a small molecule.
In this case, the small molecule is theophylline, and the rationale for using it as a target
for RNA binding was that it is a naturally occurring alkaloid that is used as a
bronchodilator. Its structure is very close to that of caffeine, and monoclonal antibodies
directed against theophylline show caffeine cross-reactivities of 0.2-0.3% of the
theophylline signal.
Can RNA also discriminate between these closely related compounds?
So - theophylline. (Jenison et al., 1994. Science 263: 1425-1428.)
The RNA pool was made of about 1013 molecules containing a 40-nucleotide random
region. [Does this represent complete sampling of sequence space for a 40 nt random
sequence? Do the arithmetic: 440 = 1024 molecules]
The protocol was to immobilize theophylline on a solid matrix. The RNA pool was applied
to the column, and allowed to mix for some time, at some temperature. This corresponds
to the ‘interaction step’ of the process.
To remove the RNA, the column was washed, first with buffer to remove unbound RNA.
Then to remove bound RNA, the column buffer containing free theophylline was applied,
to competitively bind to the RNAs and elute them as a complex. This is the ‘partitioning’
phase of the process.
To increase specificity of selection, a counter-selection was used. Buffer containing
caffeine was applied to the column, prior to addition of theophylline-buffer. Any RNA that
bound to caffeine was thus removed from the bound pool.
After eight rounds, most of the molecules in the pool bound to the column. They were
collected and sequenced. The family of RNAs contained two sequences of six and nine
nucleotides, separated by a variable span.
They could all be folded into a similar conformation.
Class exercise:
Pencil and paper, fold these two RNAs into a secondary structure.
The structure of the RNA bound to theo was solved by NMR
(Zimmerman et al., 1997. Nat Str Biol 4:644-649)