Molecular-recognition characteristics of SAM-binding riboswitches

Article abstract of DOI:10.1002/anie.200503198

(Figure Presented) A series of analogues of S-adenosylmethionine (SAM) have been used to examine the molecular-recognition characteristics of an RNA genetic element or riboswitch. Biochemical assays indicate that the riboswitch of the SAM-I class is highly selective for SAM and exhibits a pattern of molecular recognition that is distinct from that of the SAM-II class of riboswitches.

Full text of DOI:10.1002/anie.200503198

Communications
RNA
DOI: 10.1002/anie.200503198
Molecular-Recognition Characteristics of
SAM-Binding Riboswitches**
Jinsoo Lim, Wade C. Winkler, Shingo Nakamura,
Valerie Scott, and Ronald R. Breaker*
Riboswitches are highly structured domains that typically
reside within noncoding regions of certain messenger RNAs
(mRNAs).[1] These RNAs are genetic control elements that
monitor the level of specific metabolites by selective binding
without the need for intermediary protein factors. Metabolite
binding controls gene expression by several different mech-
anisms that involve allosteric changes in RNA folding.
Riboswitches typically consist of two functional domains, an
aptamer domain and an expression platform, which respec-
tively bind metabolite and trigger gene-expression changes. A
number of riboswitch classes have been discovered that
recognize a variety of important compounds, such as coen-
zymes, amino acids, and nucleobases, thus indicating that
these metabolite-sensing RNAs serve as important compo-
nents of genetic control circuitry in some organisms.[2] An
understanding of the interactions between riboswitches and
their metabolite ligands should provide greater insight into
the capabilities of RNA as a genetic switch. Furthermore, this
knowledge might empower future efforts to design novel
antimetabolites that bind to riboswitches, inhibit the expres-
sion of key metabolic genes, and thus be selectively toxic to
bacteria that carry these RNAs.[3]
S-adenosyl-l-methionine (SAM; 1; Figure 1) is an impor-
tant enzyme cofactor in many biosynthetic pathways.[4] In
bacteria of the Bacillus/Clostridium group, SAM-binding
riboswitches (SAM-I; originally termed “S-box” RNAs)[5]
control multiple different genes involved in SAMbiosynthe-
[*] Dr. J. Lim, V. Scott, Prof. R. R. Breaker
Department ofMolecular, Cellular, and Developmental Biology
Yale University
New Haven, CT 06520 (USA)
Fax: (+1)203-432-0753
E-mail: ronald.breaker@yale.edu
Dr. W. C. Winkler
Department ofBiochemistry
University ofTexas Southwestern Medical Center at Dallas
Dallas, TX 75390 (USA)
Dr. S. Nakamura
Takeda Pharmaceutical Co. Ltd.
Chuo-ku, Osaka 540-8645 (Japan)
[**] We thank Prof. G. Michael Blackburn (University of Sheffield) for
generous gifts of compounds 5 and 6 and Dr. Gail Emilsson and
other members of the Breaker laboratory for helpful comments. This
study was supported by the grants from the US National Institutes
ofHealth, the US National Science Foundation, DARPA, and the
David and Lucile Packard Foundation. SAM=S-adenosyl-
methionine.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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To uncover determinants of molecular recognition for
these RNAs, a series of thioether-modified SAManalogues
were tested for their ability to serve as ligands (Figure 1).[8]
Values for the dissociation constant (Kd) were determined by
conducting in-line probing assays[9,10] in the presence of
various concentrations of ligand (Figure 2). These assays can
be used to establish the apparent dissociation constant for
RNA–ligand complexes. As reported earlier,[6c] 2, which lacks
both a methyl group and the positive charge at the sulfur
atom, loses affinity for the SAM-I motif by a factor of 80. In
contrast, sulfoxide 3 and sulfone 4 derivatives,[11] although
lacking the methyl group, are far better ligands for SAM-I
relative to 2. For example, SAMis bound by the SAM-I motif
only sixfold better than 4. The aza analogue[12] of SAM( 5) is
more isosteric but largely uncharged under in-line probing-
assay conditions[13] and therefore is more similar to 2.
Furthermore, sinefungin 7, a SAMisoster with the same
chirality as SAM, drops by more than two orders of
magnitude in affinity. The most potent ligand for SAM-I is
an aza analogue[12] of SAM( 6) that carries an extra methyl
group and a higher positive charge density.
We conclude that SAM-I RNAs are unlikely to require
the methyl group or a specific stereochemistry of the
thioether for binding. Moreover, the net positive charge
density at the sulfur atom appears to be a key recognition
determinant for SAM-I RNAs. This position might form an
electrostatic interaction with an anionic or electronegative
group of the RNA.
This charge-density correlation is supported by the
chemical shifts of both H5’ and Hg in the 1H NMR spectrum
(Figure 3). As the thioether region is located in between H5’
and Hg, the alteration of electron density at this position
influences the chemical shifts of the protons.[12,14] When
compared with 2, the chemical shift of ribose proton H5’ for 1,
4, and 6 shifts downfield from d = 0.94 to 0.98 ppm, thus
suggesting that the net charge densities of these analogues are
highly positive.[15] The extent of the difference in the chemical
shift of the Hg proton for the derivatives is also similar to that
of H5’ (from d = 0.91 to 1.00 ppm).
The SAM-II motif exclusively binds SAM in contrast to
SAM-I and rejects even those compounds that are most
tightly bound by SAM-I, such as 4 and 6.[7] This behavior
suggests that the positive charge and the methyl group of
SAMmight both be critical for molecular recognition by
SAM-II RNAs. Moreover, the dramatic difference in the
binding affinity of 6 between the two aptamer classes implies
that the extra methyl group sterically interferes with binding
such that other contacts that exist between SAM-II and its
ligand are offset.
Subsequent to the discovery of the potent activity of 4, we
further synthesized[8] a complete set of analogues of 4, in
which one of the heteroatoms is systematically eliminated or
altered to a carbon atom while the remaining functional
groups are kept intact. The SAManalogues were prepared
from 5’-chloro-5’-deoxyadenosine derivatives,[16] which were
converted into S-adenosyl-l-homocysteine analogues by the
reaction with the l-homocysteine disodium salt.[17] Oxidation
of S-adenosyl-l-homocysteine analogues with a molybdate
Figure 1. Consensus sequence and structure for a) aptamers of SAM-I
riboswitches found in Gram-positive bacteria and b) aptamers of the
SAM-II riboswitches found in alpha proteobacteria. The black and gray
letters denote nucleotides that are conserved in 80 and 95%,
[6,7]
respectively, ofthe representatives identiifed.
c) Molecular recogni-
tion ofSAM and its analogues by the SAM-I and -II motisf.
sis, amino acid biosynthesis, and the general metabolism of
sulfur.[6] More recently,[7] we have identified a second class of
SAM-dependent riboswitches (SAM-II) in alpha proteobac-
teria. Representatives of this riboswitch class have sequence
and structural elements that are distinct from SAM-I
riboswitches even though they control genes involved in
similar metabolic processes. The existence of two distinct
riboswitch classes for SAMprovides the first opportunity to
compare the molecular-recognition capabilities of two natural
aptamers for the same ligand.
Herein, we established the molecular-recognition charac-
teristics of the aptamer domain of a SAM-I riboswitch from
the yitJ gene of Bacillus subtilis.[6c] This data is compared with
the molecular-recognition characteristics of a SAM-II ribos-
witch aptamer from the metA gene of Agrobacterium
tumefaciens.[7] We conclude from these results that the two
RNAs indeed form distinct binding pockets for SAM.
However, both classes of SAMriboswitches strongly discrim-
inate against most analogues of SAM, which is a requisite for
any metabolite-binding receptor that serves as a precision
gene-control element.
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Communications
solution containing hydrogen peroxide yielded
8–18 (Table 1).[11]
a)
b)
U
A
G
C
As SAM-II rejects 4, all the synthesized
sulfone analogues were not expected to bind
significantly to the SAM-II motif, and thus they
were tested only with SAM-I. The amino acid
modified analogues, devoid of amine or car-
boxylic acid groups (9 and 10, respectively),
had no detectable binding to SAM-I, thus
implying that the amino acid might form strong
electrostatic or hydrogen-bonding interactions
with the RNA. By examining sugar-modified
analogues, the 3’-hydroxy group appears to be
less important than the 2’-hydroxy group (13
and 12, respectively). The 6-amino group of
adenine is so important that replacement by
either a hydroxy group or a hydrogen atom (15
and 14, respectively) is not tolerated. The 7-aza
group is likely to be critical for hydrogen
bonding, whereas the 3-aza group is less
important than any other amine in the adenine
ring (16 and 17, respectively). An extra amino
group at the 2-position of adenine (18)
decreases binding by a factor of 67, thus
suggesting a tight pocket is formed in this case.
In summary, we have examined and com-
pared the molecular-recognition characteristics
of both the SAM-I and -II aptamers
(Scheme 1) by using in-line probing assays
with a series of SAManalogues. The 6-amino,
7-aza, and 2’-hydroxy groups of the nucleobase,
and the functional groups on the amino acid
moiety of SAMappear to participate in
molecular recognition with SAM-I. However,
SAM-II is far more selective for the thioether
linkage of SAM, and therefore a different
analogue set was used to further establish other
molecular determinants for this RNA.[7] The
fact that each riboswitch has different sub-
strate-recognition determinants confirms that
their sequence and structural differences form
distinct binding pockets for SAM. Further-
more, there appears to be considerable oppor-
tunity to design novel SAManalogues that
selectively trigger SAM-I riboswitches, perhaps
without risk of interacting with other SAM-
binding biopolymers. Given that SAM-I ribos-
witches in Gram-positive organisms control
numerous genes whose products participate in
fundamental metabolic pathways, such SAM
analogues might find application as antibiotic
compounds that selectively inhibit bacterial
growth.
A
C
G
C
C
G
G
U
A
A
U
122
108
A
G118
A114
A110
U
G
124 yitJ
aptamer
G
U
A
C
100
80
G
G
C
C
A
CA
99
89
A
G
G
U
P3
A
C
G
C
U
60
G
81
72
A
G
A
G
C C C
A
G
A
A
A
A
A
A
G
A
G
C U U C
G A A
U
G
A75
A74
C
U
C
G G G
P2a
C
C
A
A
A
G
C
G
G
P2
120
A A G C U
40
A
G
A
C
G
A
A
G U U C G
A
A
C
Constant scission
Reduced scission
Increased scission
C
U
G
A
P4
60
50
140
A
U
U
C
U
U
U
A
P1
A
G
A
A
G
28
G
5′
A G
3′
ppp * *
GG
A40
G39
39
1
0.75
0.50
0.25
0
c)
NH2
N
N
N
N
O
G39/A40
A74/A75
A110
S
O
O
+
NH
3
4
A114
HO OH
G118
–10
–9
10
–8
10
–7
10
–6
10
–5
10
–4
10
–3
10
10
analogue / M
Figure 2. SAM-I riboswitches and a representative in-line probing assay using 124 yitJ RNA from
B. subtilis and various concentrations ofSAM. a) Sequence and secondary-structure model ofthe
yitJ SAM-I aptamer. P1–P4 identify conserved base-pairing elements. Unshaded, encircled
nucleotides indicate a putative pseudo knot interaction. Nucleotide 1 corresponds to the putative
transcriptional start site. Asterisks identify nucleotides that were added to the construct to permit
efficient transcription in vitro. The first nucleotide of the AUG start codon is 235 (not shown).
Sites ofstructural modulation upon introduction ofSAM are depicted as described. b) Sponta-
neous cleavage patterns of124 yitJ in the absence (À) or presence ofSAM at the molar
concentrations indicated were examined after separation by polyacrylamide gel electrophoresis.
NR=no reaction, T1=partial digest with RNase T1, ÀOH=partial digest with alkali, respectively.
Certain fragment bands corresponding to digestion by T1 (which cleaves after guanine residues)
are depicted. Bars identify positions of substantial modulation of spontaneous cleavage that were
used for quantification (see the Experimental Section). c) Plot of the normalized fraction of
124 yitJ RNA cleaved at each site designated versus the concentration ofthe sulof ne analogue
ofSAM. Hal-fmaximum change in spontaneous cleavage occurs at a concentration of 4 of
approximately 50 nm, which reflects the apparent Kd value ofthis ligand with the yitJ aptamer.
4
Experimental Section
The aptamer domain from the SAM-I riboswitch of
the yitJ gene from B. subtilis (Figure 2a) was gen-
erated as described previously.[6c] 5’-32P-Labeled
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Scheme 1. Predicted molecular-recognition determinants ofSAM ribo-
switches. The schematic representation reflects a possible pattern of
molecular interactions between SAM and a) the aptamer domain of
SAM-I riboswitches and b) the aptamer domain ofSAM-II ribo-
switches. Note that, in some cases, the precise type ofmolecular
contact or the number of contacts for a given functional group cannot
be determined from the limited data currently available. Shading on
the hydrocarbon side chain ofthe methionyl moiety relfects the
importance oflength. Also the molecular-recognition patterns are likely
incomplete for both aptamer classes. A more comprehensive assess-
ment ofthe contacts between SAM-II RNA and its ligand will require
the use ofanalogues that retain the natural methylated thioether
linkage, and the design oftight-binding analogues would be greatly
aided by an atomic-resolution structure ofthe RNA bound to SAM.
Figure 3. Charge-density correlation plot. Binding affinities (Kd) of
analogues are inversely proportional to the chemical shifts of the
protons H5’ (diamonds) and Hg (squares) ofthe corresponding
derivatives.
RNA was prepared using calf intestinal phosphatase (Roche Diag-
nostics), T4 polynucleotide kinase (New England Biolabs), and
[g-32P]ATP (Amersham Biosciences) according to the manufacturerꢀs
directions and purified by polyacrylamide gel electrophoresis. Sim-
ilarly, radiolabeled RNAs that represent the SAM-II riboswitch
aptamer for the metA gene from A. tumefaciens were prepared.
Genomic DNA used to prepare the template for transcription of the
SAM-II construct[7] was obtained from Agrobacterium tumefaciens
strain GV2260 (a gift from the S. Dinesh-Kumar Lab, Yale
University). For each probing reaction, approximately 1 nm 5’-32P-
labeled RNAwas incubated for about 40 h at 258C in 50 mm Tris–HCl
(pH 8.3 at 258C), 20 mm MgCl2, 100 mm KCl, with added compounds
as indicated for each experiment. Additional experimental details are
similar to those described elsewhere.[2a–b] Polyacrylamide gels that
separate the RNA cleavage products from in-line probing assays were
scanned and quantitated using a molecular dynamics STORM820
PhosphorImager system. The Kd value of 124 yitJ for SAMand for
each SAManalogue was determined by plotting the normalized
fraction of RNA cleaved at six regions (e.g., Figure 2c) versus the
logarithm of the concentration of SAMin molar units. The concen-
tration of SAMneeded to induce half-maximum change in RNA-
cleavage extent was interpreted as the apparent Kd value. Similar data
for the SAM-II 164 metA RNA is reported elsewhere.[7]
Received: September 8, 2005
Published online: December 28, 2005
Table 1: Dissociation constant (Kd) values for SAM analogues with the yitJ SAM-I riboswitch aptamer from B. subtilis.
R1
R2
R3
H
R4
R5
W
X
Y
Z
Kd [mm]
4
NH2
CO2H
OH
OH
NH2
N
N
H
3”10À2
8
9
H
H
CH2OH
CO2H
H
H
H
H
OH
H
H
H
H
H
H
H
OH
OH
OH
H
OH
OH
OH
OH
OH
H
OH
OH
OH
OH
OH
NH2
NH2
NH2
NH2
NH2
NH2
H
N
N
N
N
N
N
N
N
CH
N
N
N
N
N
N
N
N
N
N
N
CH
N
H
H
H
H
H
H
H
H
>1000
>1000
>1000
40
30
5
>1000
>1000
>1000
3
10
11
12
13
14
15
16
17
18
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
H
OH
OH
OH
OH
OH
OH
OH
NH2
NH2
NH2
H
H
NH2
2
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Communications
Keywords: aptamers · molecular recognition · riboswitches ·
.
RNA · structure–activity relationships
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1
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