INSIGHT
SINGLE-MOLECULE BIOPHYSICS
The light side of the force
A combination of two single-molecule techniques has revealed new
tertiary interactions in the TPP riboswitch.
AAKASH BASU AND TAEKJIP HA
Related research article Duesterberg VK,
Fischer-Hwang IT, Perez CF, Hogan DW, Block
SM. 2015. Observation of long-range tertiary
interactions during ligand binding by the TPP
riboswitch aptamer. eLife 4:e12362. doi: 10.
7554/eLife.12362
Image Schematic of the experimental setup
used to study the TPP riboswitch.
S
Copyright Basu and Ha. This
article is distributed under the terms
of the Creative Commons Attribution
License, which permits unrestricted
use and redistribution provided that
the original author and source are
credited.
ingle-molecule techniques are widely
used to investigate how the structure of
a molecule changes over time. Such
measurements have advantages over standard
biochemical experiments, which can only record
average values measured over a large number of
molecules, and traditional structural biology
techniques that cannot gather dynamic information. However, a drawback of single-molecule
techniques is that they typically involve measuring how just a few structural parameters – often
only one, such as the end-to-end length of the
molecule – vary over time. Thus, a growing trend
in the field is to use hybrid instruments that combine different single-molecule techniques, and
so allow more than one parameter to be
observed simultaneously (Ha, 2014).
Riboswitches are regions in the non-coding
regions of messenger RNA (mRNA) molecules
that control the expression of the mRNA by
sensing and binding to specific small ’ligand’
molecules. Riboswitches comprise an aptamer
region that undergoes structural transitions
Basu and Ha. eLife 2016;5:e14274. DOI: 10.7554/eLife.14274
when a ligand binds to it, and an expression
platform that regulates the expression of the
mRNA in response to these shape changes.
Now, in eLife, Steven Block and co-workers from
Stanford University – Van Duesterberg, Irena
Fischer-Hwang, Christian Perez and Daniel
Hogan – combine two single-molecule techniques to investigate the structural changes that
occur in the thiamine pyrophosphate (TPP)
riboswitch as it unfolds and binds to TPP and
other ligands (Duesterberg et al., 2015).
Most riboswitches are present only in bacteria, but the TPP riboswitch is present in all kingdoms of life. When bound to a molecule of TPP,
the structure of this riboswitch resembles an
inverted ‘h’ (Figure 1), with TPP docked in the
region between the two ’sensor arms’ of the
aptamer.
Force-extension curves can be obtained by
stretching individual riboswitches and measuring
the end-to-end length of the molecule
(Greenleaf et al., 2008). This technique has
been used to monitor the mechanical unfolding
of secondary structures in the TPP riboswitch,
such as the various helixes that make up the
aptamer (Anthony et al., 2012). Depending on
how the TPP molecule is bound to the riboswitch
(it can be bound strongly, weakly or not at all),
these secondary structures unfold at different
values of pulling force, thus producing distinct
force-extension curves. However, these curves
are typically insensitive to long-range tertiary
interactions, such as the interactions that occur
between the L5 structural loop at the tip of one
sensor arm and the P3 helix at the end of the
other arm (Figure 1).
Additionally, the pattern of secondary structure unfolding of the weakly bound form of the
riboswitch does not change when TPP is
replaced
with
thiamine
monophosphate
1 of 3
Insight
Single-Molecule Biophysics The light side of the force
Figure 1. Watching the TPP riboswitch unfold. The ligand TPP (yellow) binds to the riboswitch in the region
between the two sensor arms of its aptamer, and also makes contact with each arm. There is also a long-range
tertiary interaction between the loop L5 at the tip of one arm and the helix P3 in the other arm. Duesterberg,
Fischer-Hwang, Perez et al. used optical trapping to apply a pulling force to the ends of the molecule and to
measure the overall end-to-end extension (Dx). Simultaneously, they used smFRET to measure the separation
between fluorophores (green and red fuzzy circles) placed on L5 and P3. Mechanical unfolding and the state of
ligand binding affect both secondary structures and tertiary interactions. Secondary structural changes, such as
unzipping one or more helices (helix P1 in this example), cause a corresponding change in Dx. Changes in longrange tertiary structure, such as the two sensor arms docking with each other, does not affect Dx, but cause a
corresponding change in FRET efficiency.
(TMP, which contains one phosphate group,
rather than the two found in TPP) or thiamine
(which contains no phosphate groups). This
leaves open the possibility that the phosphates
alter tertiary interactions, as these changes are
undetectable
in
force-extension
curve
measurements.
A technique called smFRET has been used
extensively to investigate how tertiary structures
change in large molecular complexes and, more
recently, in riboswitches (Lemay et al., 2006).
Now, Duesterberg, Fischer-Hwang, Perez et al.
have used a combined force-smFRET approach
(Hohng et al., 2007; Tarsa et al., 2007) to
stretch the TPP riboswitch complex while simultaneously observing both the end-to-end extension and changes in long-range tertiary
interactions. smFRET measures the latter by
monitoring the energy transfer between two fluorophores placed on the P3 helix and the L5
loop; the smFRET signal depends on the distance between these structural elements.
Duesterberg et al. tested TPP riboswitches
that were weakly bound to one of TPP, TMP or
thiamine. All three ligands showed the same pattern of force-induced unfolding of secondary
structures. However, the smFRET results indicated that P3 and L5 were further apart when
Basu and Ha. eLife 2016;5:e14274. DOI: 10.7554/eLife.14274
the riboswitch was bound to TMP or thiamine.
Thus, each additional phosphate group seems to
draw the sensor arms of the riboswitch closer
together during ligand binding.
Further, although the two arms are more separated in the presence of bound TMP or thiamine, in all cases there was some variability in
the separation distance between the arms. However, this variability is greater in the TPP-bound
case. Duesterberg et al. hypothesize that the
flexibility of the riboswitch’s structure when
weakly bound to TPP might be essential for
proper docking of the helix arms and the subsequent transition of the aptamer to the strongly
bound configuration.
Both smFRET and force-extension curves
have widely been used to study the folding of
large biological molecules. However, this work
by Duesterberg et al. represents the first example of combining the two in order to simultaneously report on secondary and tertiary
structural changes during folding and ligand
binding. Large-scale tertiary structural transitions
are ubiquitous in biology. Applying similar
approaches to the wide body of proteins and
RNAs whose folding characteristics have been
studied by force-extension curves alone will
greatly enhance our understanding of the overall
2 of 3
Insight
Single-Molecule Biophysics The light side of the force
structural transitions essential for their biological
functions.
The relative ease of implementation of
smFRET detection has made it a technique of
choice for combining with other single-molecule
techniques. For instance, FRET has recently
been combined with patch clamping in order to
correlate structural transitions in ion channels
with the flow of ionic current (Sasmal and Lu,
2014). Combining traditional single-molecule
workhorses, as well as pairing single-molecule
techniques with other recent advances in coarsegrained modeling and genomic techniques, will
likely serve the critical need to increase the number of observable parameters available when
studying molecules one at a time.
Aakash Basu is in the Department of Biophysics and
Biophysical Chemistry, Johns Hopkins University,
Baltimore, United States
Taekjip Ha is in the Department of Biophysics and
Biophysical Chemistry, the Department of Biomedical
Engineering and the Howard Hughes Medical Institute,
Johns Hopkins University, Baltimore, United States
[email protected]
Competing interests: The authors declare that no
competing interests exist.
Published 23 February 2016
pyrophosphate riboswitch aptamer. Proceedings of
the National Academy of Sciences USA 109:1485–
1489. doi: 10.1073/pnas.1115045109
Duesterberg VK, Fischer-Hwang IT, Perez CF, Hogan
DW, Block SM. 2015. Observation of long-range
tertiary interactions during ligand binding by the TPP
riboswitch aptamer. eLife 4:e12362. doi: 10.7554/
eLife.12362
Greenleaf WJ, Frieda KL, Foster DA, Woodside MT,
Block SM. 2008. Direct observation of hierarchical
folding in single riboswitch aptamers. Science 319:
630–633. doi: 10.1126/science.1151298
Ha T. 2014. Single-molecule methods leap ahead.
Nature Methods 11:1015–1018. doi: 10.1038/nmeth.
3107
Hohng S, Zhou R, Nahas MK, Yu J, Schulten K, Lilley
DM, Ha T. 2007. Fluorescence-force spectroscopy
maps two-dimensional reaction landscape of the
Holliday junction. Science 318:279–283. doi: 10.1126/
science.1146113
Lemay JF, Penedo JC, Tremblay R, Lilley DM,
Lafontaine DA. 2006. Folding of the adenine
riboswitch. Chemistry & Biology 13:857–868. doi: 10.
1016/j.chembiol.2006.06.010
Sasmal DK, Lu HP. 2014. Single-molecule patch-clamp
FRET microscopy studies of NMDA receptor ion
channel dynamics in living cells: revealing the multiple
conformational states associated with a channel at its
electrical off state. Journal of the American Chemical
Society 136:12998–13005. doi: 10.1021/ja506231j
Tarsa PB, Brau RR, Barch M, Ferrer JM, Freyzon Y,
Matsudaira P, Lang MJ. 2007. Detecting force-induced
molecular transitions with fluorescence resonant
energy transfer. Angewandte Chemie International
Edition 46:1999–2001. doi: 10.1002/anie.200604546
References
Anthony PC, Perez CF, Garcia-Garcia C, Block SM.
2012. Folding energy landscape of the thiamine
Basu and Ha. eLife 2016;5:e14274. DOI: 10.7554/eLife.14274
3 of 3