Nelson et al. and Breaker c-di-AMP Riboswitches Supplementary Information Riboswitches in eubacteria sense the second messenger c-di-AMP James W. Nelson 1 , Narasimhan Sudarsan 2,3 , Kazuhiro Furukawa 3,* , Zasha Weinberg 2,3 , Joy X. Wang 3,** , Ronald R. Breaker 2,3,4 † 1 Department of Chemistry, Yale University, Box 208107, New Haven, CT 06520, USA 2 Howard Hughes Medical Institute, 3 Department of Molecular, Cellular and Developmental Biology, Yale University, Box 208103, New Haven, CT 06520, USA. 4 Department of Molecular Biophysics and Biochemistry, Yale University, Box 208103, New Haven, CT 06520, USA. Current addresses: *Faculty of Pharmaceutical Sciences, The University of Tokushima, Tokushima, Japan; **Center for Clinical and Translational Metagenomics, Brigham and Women's Hospital, Boston, MA 02115 † To whom correspondence should be addressed. E. mail: [email protected]Dr. Ronald R. Breaker Tel: (203) 432-9389 Fax: (203) 432-0753 E-mail: [email protected]Nature Chemical Biology: doi:10.1038/nchembio.1363
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Nelson et al. and Breaker c-di-AMP Riboswitches
Supplementary Information
Riboswitches in eubacteria sense the
second messenger c-di-AMP
James W. Nelson1, Narasimhan Sudarsan
2,3, Kazuhiro Furukawa
3,*, Zasha
Weinberg2,3
, Joy X. Wang3,**
, Ronald R. Breaker2,3,4
†
1Department of Chemistry, Yale University, Box 208107, New Haven, CT 06520,
USA 2Howard Hughes Medical Institute,
3Department of Molecular, Cellular and
Developmental Biology, Yale University, Box 208103, New Haven, CT 06520, USA. 4Department of Molecular Biophysics and Biochemistry, Yale University, Box
208103, New Haven, CT 06520, USA.
Current addresses: *Faculty of Pharmaceutical Sciences, The University of
Tokushima, Tokushima, Japan; **Center for Clinical and Translational
Metagenomics, Brigham and Women's Hospital, Boston, MA 02115
† To whom correspondence should be addressed. E. mail: [email protected]
Supplementary Figure 1│In-line probing of 165 ydaO RNA with yeast extract. a,
Pattern of spontaneous cleavage resulting from equilibrium dialysis/in-line probing of the
B. subtilis 165 ydaO RNA in the absence of any added compounds or in the presence of
specific dilutions of yeast extract. Annotations, including the sites of structural
modulation, are as described in the legend for Fig. 1d. b, Mass spectrometry of an HPLC
fraction that induces 165 ydaO RNA conformational change (active fraction) and an
adjacent fraction that does not induce conformational change (inactive fraction). The
most prominent peak unique to the active fraction (arrow) corresponds to the mass (m/z =
346.0554 for the [M-H]- ion) of adenosine monophosphate. MS/MS analysis (data not
shown) further suggested that this compound was either adenosine 5′ monophosphate, or
one of its close structural isomers (adenosine 2′- or 3′-monophosphate). Given our
previous examination and exclusion of 5´ AMP as a biologically relevant ligand for ydaO
RNAs5, we hypothesized that the natural ligand included an AMP moiety that was not
stable under the ionization conditions used for mass spectrometry, leading us to test a
wide variety of potential ligand candidates (Supplementary Fig. 2), including c-di-AMP.
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Nelson et al. and Breaker c-di-AMP Riboswitches
Supplementary Figure 2│Screening for analogs of 5´ AMP that are bound by the B.
subtilis 165 ydaO RNA. Depicted are the regions of several in-line probing gels
corresponding to the sites of modulation denoted 1 and 2 as described in Fig. 1d. The
concentrations used for each compound in the box are 0.01, 0.1 and 1 mM, and these
results are compared to that for c-di-AMP at 0.01 mM. 5´ IMP denotes inosine-5´-
monophosphate and A 5´PS denotes adenosyl-5´-phosphosulfate. Other annotations are
as described in the legend to Fig. 1d.
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Nelson et al. and Breaker c-di-AMP Riboswitches
Supplementary Figure 3│In-line probing of the 165 ydaO RNA in the absence and
presence of 100 nM c-di-AMP. This data (see the legend to Fig. 1 for details) was used
in conjunction with the data in Fig. 1d to map the sites of spontaneous cleavage. Note
that some sites of spontaneous cleavage (occurring 3´ of the nucleotide identified) can
only be estimated due to low resolution of the gels (particularly sites closer to the 3´
terminus of the construct).
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Nelson et al. and Breaker c-di-AMP Riboswitches
Supplementary Figure 4 (Part 1)│Ligand binding by the yuaA RNA from B. subtilis. a, PAGE analysis in-line probing reactions using 150 yuaA RNA from B. subtilis exposed
to various concentrations of c-di-AMP (100 pM to 10 μM) or ATP (17.8 μM to 3.16
mM). Methods and annotations are as described for Fig. 1d.
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Nelson et al. and Breaker c-di-AMP Riboswitches
Supplementary Figure 4 (Part 2)│b, Sequence, secondary structure, and structural
modulation of the 150 yuaA RNA from B. subtilis. Locations were mapped using the data
in a. c, Plot of the fraction of riboswitch RNA bound to ligand versus the logarithm of the
molar concentration of c-di-AMP as inferred from the modulation of spontaneous
cleavage products in a. The KD value of this riboswitch for c-di-AMP is ~450 pM, and
the curve is consistent with that expected for a one-to-one binding interaction between
RNA and ligand.
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Supplementary Figure 5│Examining the KD for the 165 ydaO construct by in-line
probing using an RNA concentration near the lower limit of detection (~0.1 nM). a,
PAGE analysis of an in-line probing assay with 165 ydaO RNA from B. subtilis exposed
to various concentrations of c-di-AMP (5 pM to 100 nM). Methods and annotations are
as described for Fig. 1d. b, Plot of the fraction of riboswitch RNA bound to ligand versus
the logarithm of the molar concentration of c-di-AMP as inferred from the modulation of
spontaneous cleavage products in a. Note that half maximal modulation occurs at ~100
pM but that the curve remains steeper than that expected for a 1-to-1 interaction,
suggesting that the RNA concentration remains higher than the KD for ligand binding.
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Supplementary Figure 6│In-line probing analysis of the 144 ydaO RNA construct
from B. subtilis. a, Sequence and secondary structure model for the 144 ydaO RNA from
B. subtilis. b, PAGE analysis of an in-line probing assay with 144 ydaO RNA exposed to
various concentrations of c-di-AMP (100 pM to 10 μM) or ATP (17.8 μM to 3.16 mM).
Other annotations are as described in the legend to Fig. 1d. c, Plot of the fraction of
RNAs undergoing structural modulation versus the logarithm of the concentration of c-
di-AMP. Values were derived by evaluating the bands undergoing changes in b. Error
bars are the standard deviation of the normalized fraction modulated for bands 1, 2 and 5.
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Supplementary Figure 7│Modulation of a c-di-AMP riboswitch from B. subtilis by
c-di-AMPSS, a phosphorothioate-modified analog of c-di-AMP. a, Chemical structure
of c-di-AMPSS. b, PAGE analysis of an in-line probing assay of ydaO 165 exposed to
various concentrations of c-di-AMPSS (100 pM to 100 μM). Annotations are as described
in the legend to Fig. 1d. c, Plot of the fraction of RNAs undergoing structural modulation
versus the logarithm of the concentration of c-di-AMPSS. Values were derived by
evaluating the bands undergoing changes in b. Note that, although the calculated KD is
approximately 7 nM, this is near the expected concentration of RNA in the reaction tubes.
Therefore, the actual KD is most likely lower than this value.
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Supplementary Figure 8│Evidence of preferential c-di-AMP binding by a ydaO
RNA representative from Nostoc punctiforme. a, PAGE analysis of an in-line probing
assay with 139 ydaO RNA from Nostoc punctiforme that was exposed to various
concentrations of c-di-AMP or to 1 mM ATP. Vertical lines identify in-line probing
products whose amounts change on addition of c-di-AMP. Methods and other annotations
are as described for Fig. 1d. b, Sequence, secondary structure, and structural modulation
of the 139 ydaO RNA from N. punctiforme. Locations were mapped using the data in a.
c, Plot of the fraction of riboswitch RNA bound to ligand versus the logarithm of the
molar concentration of c-di-AMP as inferred from the modulation of spontaneous
cleavage products in a. The KD value of this riboswitch for c-di-AMP is ~30 nM, and the
curve depicted is that expected for a one-to-one binding interaction between RNA and
ligand.
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Supplementary Figure 9│Evidence of preferential c-di-AMP binding by a ydaO
RNA representative from Syntrophus aciditrophicus. a, PAGE analysis of an in-line
probing assay with 137 ydaO RNA from Syntrophus aciditrophicus exposed to various
concentrations of c-di-AMP or 1 mM ATP. Methods and annotations are as described for
Supplementary Fig. 8. b, Sequence, secondary structure, and structural modulation of
the 137 ydaO RNA from S. aciditrophicus. Locations were mapped using the data in a. c,
Plot of the fraction of riboswitch RNA bound to ligand versus the logarithm of the molar
concentration of c-di-AMP as inferred from the modulation of spontaneous cleavage
products in a. The KD value of this riboswitch for c-di-AMP is ~550 pM, and the curve
depicted is that expected for a one-to-one binding interaction between RNA and ligand.
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Supplementary Figure 10│Evidence of preferential c-di-AMP binding by a ydaO
RNA representative from Clostridium acetobutylicum. a, PAGE analysis of an in-line
probing assay with 130 ydaO RNA from Clostridium acetobutylicum exposed to various
concentrations of c-di-AMP or 1 mM ATP. Methods and annotations are as described for
Supplementary Fig. 8.b, Sequence, secondary structure, and structural modulation of the
130 ydaO RNA from C. acetobutylicum. Locations were mapped using the data in a. c,
Plot of the fraction of riboswitch RNA bound to ligand versus the logarithm of the molar
concentration of c-di-AMP as inferred from the modulation of spontaneous cleavage
products in a. The KD value of this riboswitch for c-di-AMP is ~1 nM, and the curve
depicted is that expected for a one-to-one binding interaction between RNA and ligand.
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Supplementary Figure 11│In-line probing analysis of the 165 ydaO RNA construct
from B. subtilis under sub-optimal conditions. a, PAGE analysis of an in-line probing
assay with ydaO RNA from B. subtilis exposed to various concentrations of c-di-AMP
and ATP with 10 mM MgCl2 and at 37 °C for 16 hours. Methods and other annotations
are as described for Fig. 1d. b, Plot of the fraction of riboswitch RNA bound to ligand
versus the logarithm of the molar concentration of c-di-AMP as inferred from the
modulation of spontaneous cleavage products in a at sites 1, 2, and 3. Not unexpectedly,
the KD of c-di-AMP has increased relative to that obtained under standard in-line probing
conditions to 10 nM. Importantly, however, no consistent modulation with ATP is
observed at concentrations even as high as 5 mM.
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Supplementary Figure 12│Riboswitch-mediated gene expression with constructs
driven by different promoters. a, Construct design wherein the promoter (P) is either
the native promoter for the ydaO gene, or the promoter for the B. subtilis lysC gene. b,
Predicted intrinsic transcription terminator stem for the B. subtilis ydaO riboswitch and
its fusion to the lacZ reporter gene. c, Plot of the amount of reporter gene (see b)
expression observed in B. subtilis YP79 cells carrying a knock-out of the disA gene,
normalized to the level observed with unaltered B. subtilis cells, grown in lysogeny broth.
Error bars are the standard deviation of three independent measurements.
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Supplementary Figure 13 (previous page)│Genomic locations of c-di-AMP
riboswitches in several organisms. Each riboswitch occurs singularly (denoted by hairpin
annotation), but in some instances appears to control the expression of several genes in an
operon. For representative 3 in B. anthracis, the gene with the asterisk indicates the
presence of an unannotated pseudogene with sequence similarity to a portion of potE (a
putrescine transporter). This possible gene is followed by a large intergenic region and
then the gene for hemolytic enterotoxin HBL (not depicted). The gene name for yuaA
(now ktrA) has been updated in response to recent protein function studies61
, while ydaO
has been renamed potE due to its similarity to this gene as determined by Protein-
BLAST.
The riboswitch locations in these organisms are typical of the patterns of associations
observed between c-di-AMP riboswitches and the genes they likely control among
diverse bacterial lineages. For example, c-di-AMP riboswitches almost exclusively
control genes involved in cell wall metabolism in Actinobacteria, whereas members of
this same riboswitch class in Cyanobacteria and Bacillales appear to control genes more
directly related to overcoming osmotic stress.
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Supplementary Figure 14 (previous page)│In-line probing of the 165 ydaO RNA in
increasing concentrations of c-di-AMP or ATP. Polyacrylamide gel electrophoresis
(PAGE) analysis of an in-line probing assay with 165 ydaO RNA exposed to various
concentrations of c-di-AMP (100 pM to 10 μM in half-log intervals) or ATP (17.8 μM to
3.16 mM in quarter-log intervals). NR, T1 and ‒
OH designate no reaction, partial
digestion with either RNase T1 (cleaves after guanosine nucleotides) or hydroxide ions
(cleaves after any nucleotide),. Precursor RNA (Pre) and certain RNase T1 cleavage
product bands are identified. Locations of spontaneous RNA cleavage changes brought
about by c-di-AMP (regions 1 through 6) are identified by asterisks. This is the full
length gel presented in part in Fig. 1d in the main text.
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Supplementary Figure 15 │Mutation of conserved nucleotides in the 165 ydaO RNA
abolishes c-di-AMP binding. In-line probing analyses of WT and M1 through M4
RNAs in the absence (‒ ) or presence of 10 nM c-di-AMP. This is the full length image
of the gel sections presented in Fig. 2c and Fig. 2d.
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Nelson et al. and Breaker c-di-AMP Riboswitches
Supplementary Figure 16│Deletion of the pseudoknot in 165 ydaO RNA abolishes
ATP binding and modestly reduces c-di-AMP binding. In-line probing analysis of M5
in the absence (‒ ) of ligand, or presence of increasing c-di-AMP or 1 mM ATP. The gel
images depict the region encompassing sites 1 through 6 with annotations as described
for Fig. 1d. This is the full gel presented in Fig. 2d.
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Supplementary Figure 17│In vitro transcription termination of the yuaA RNA. PAGE analysis of an in vitro transcription termination assay using the yuaA riboswitch
from B. subtilis. T is the riboswitch-terminated RNA transcript and FL is the full-length
run-off transcript. M is a marker lane comprising the transcription products from a similar
DNA template encoding the riboswitch plus six additional nucleotides beyond the
predicted terminator site. This is the full length gel presented in Fig. 3a. Note that the
additional lane at the right of the gel is not present in the cropped image, as it is simply
the same reaction presented in the marker lane.
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Nelson et al. and Breaker c-di-AMP Riboswitches
Supplementary Table 1. Category assignment of genes for protein domains
controlled by c-di-AMP riboswitches. Genes predicted to be controlled by the c-di-
AMP riboswitch were manually assigned to one of several categories listed below based
on the description of domain(s) within that gene from the conserved domain database
(see Materials and Methods). Genes that are not grouped into these categories are not
shown here, but can be found in the Supplementary Online Material file. These categories
are the basis for Fig. 4.
Category Domain
accession
Brief description (from the Conserved Domain Database)