SUPPLEMENTARY INFORMATION
For Numata et al.
Supplementary Discussion:
Implications for hypermodifications in the anticodon loop of MnmA substrates:
Position 5 of U34 of tRNAGlu, tRNALys and tRNAGln in most organisms is
hypermodified with a methylaminomethyl (mnm5), carboxymethylaminomethyl
(cmnm5), taurinomethyl (τm5) or methoxycarbonylmethyl (mcm5) moiety besides the
2-thio group1-3. In the structure of the initial tRNA binding state, the C5 atom of U34 is
accessible to the solvent (Fig. 3d), indicating that the hypermodified U34 easily binds to
the MnmA active site. In contrast, in the current pre-reaction and adenylated intermediate
forms, the C5 atom provides a van der Waals contact to the aromatic ring of Phe154 (Fig.
3e, f), which precludes the accommodation of the 5-methyl substituents. Since the
structures around Phe154 are remarkably flexible exhibiting high B-factors, a slight
structural rearrangement might allow the accommodation of mnm5U34 for sulfuration.
Thus, the 2-thio and 5-methylaminomethyl modifications might occur independently, as
described previously4,5.
The tRNA substrates of MnmA contain a modified A37 (2-methyl adenosine, m2A,
or N6-threonylcarbamoyladenosine, t6A) in their anticodon loop6. These modifications
are essential for reading frame maintenance7. In the present structures, A37 is stabilized
by base stacking with C36, and the C2 and N6 positions are exposed to the solvent (Fig.
2b), suggesting that the modified A37 can be accommodated. Furthermore,
superimposition of t6A37 on our structures showed that the N6-threonylcarbamoyl group
is located in close proximity to the conserved Arg313 in all structures. Despite the fact
that the R313A mutant only reduced the sulfuration of unmodified tRNAGlu 2 fold (Fig.
2d), the guanidino group of Arg313 might form a salt bridge with the carboxyl group of
the N6-threonylcarbamoyl moiety to stabilize the interaction between MnmA and
modified tRNA. Therefore, it can be postulated that the A37 modification precedes the
s2U34 modification.
Implication for the reaction mechanism of 2-selenouridine synthase YbbB:
In a variety of eubacteria and possibly archaea, mnm5s2U34 of tRNAGlu, tRNALys
and tRNAGln is occasionally further modified into mnm5se2U34 (Ref. 8), by replacement
of the 2-thio group with selenium9. Recently, it was found that the ybbB gene product
(SePO3-dependent 2-selenouridine synthase) catalyzes this reaction10. YbbB is likely to
utilize selenophosphate to generate a perselenide intermediate on its catalytic Cys residue
in the rhodanese domain, and the neighboring domain that contains a P-loop motif
(Walker A motif) is thought to transfer the Se atom into the anticodon wobble position of
the tRNAs10. This mechanism might be similar to that of ThiI11,12, the s4U modifying
enzyme. Therefore, although the precise function of the P-loop motif of YbbB is as yet
unknown, it is possible that the reaction mechanism of YbbB resembles that of MnmA.
Supplementary Methods:
Structure refinement.
The current model of the Form II structure contains all of the protein and tRNA
residues, except for the N-terminal three residues of MnmA and A71-A76 of tRNA, and
exhibits an Rwork of 26.5% for 95% of the data. The Rfree for the remaining 5% of the data
is 30.6%. The current model of the Form I structure contains residues 5-188 and 205-368
in A molecule of MnmA complexed with tRNA C molecule (G1-C74), and residues 4-189
and 196-368 in B molecule of MnmA complexed with tRNA D molecule (C3-G73),
exhibiting final Rwork and Rfree values of 22.6% and 27.1%, respectively, for the data
between 50 and 3.1 Å resolution. In the Form III structure, after torsion-angle simulated
annealing, the density corresponding to the adenylate moiety covalently attached to the
C2 atom of U34 was clearly visible in the resultant Fo-Fc map. Topology and parameter
files for the covalent intermediate, 2-adenylyl-uridine, at the wobble position were
generated using HIC-UP (http://xray.bmc.uu.se/hicup/). The structure of the Form III
crystals was refined to an Rwork of 28.1% and an Rfree of 31.8% at 3.4 Å resolution. The
current model of the Form III structure contains all of the protein and tRNA residues,
except for the N-terminal four residues of MnmA and C75-A76 of tRNA, in both
complexes.
Detection of the adenylated intermediate
The adenylation of the tRNA was performed, as described previously13, at 37˚C in
10 l of 50 mM Tris-HCl buffer (pH 7.6) containing 50 mM KCl, 12 mM Mg Mg(OAc)2,
1 mM DTT, and 5 M [-32P]ATP (~300 Ci/mmol, Amersham Pharmacia Biotech.). The
reaction was initiated by mixing 1 g (4 M) tRNA and 0.48 g (4 M) MnmA, and was
incubated for 30 min. The reaction was quenched by adding 10 volumes of 5% formic
acid, followed by RNA extraction with ISOGEN (Wako chemical). The RNA was
subjected to polyacrylamide gel electrophoresis under denaturing conditions. The gel was
stained with ethidium bromide (EtBr), and was also exposed on an imaging plate to
visualize the radioactivity using the FLA7000 system (Fujifilm).
In vitro sulfur incorporation analysis
Mutations were generated by the QuikChangeTM Site-Directed Mutagenesis kit
(Stratagene) according to the manufacturer’s instructions. The in vitro transcribed tRNA
was dephosphorylated by bacterial alkaline phosphatase (Takara), and was labeled at its
5’ terminus with [-32P]ATP (110 TBq/mmol) by T4 polynucleotide kinase (Toyobo). The
tRNA was then purified under denaturing conditions by 10% polyacrylamide gel
electrophoresis. The reaction was carried out in a total volume of 20 l containing 50 mM
Tris-HCl buffer (pH 7.5), 50 mM KCl, 12 mM Mg(Oac)2, 20 M pyridoxal 5’-phosphate,
2 mM ATP, 0.1 mM DTT, 0.1 mM cysteine, 0.19 g (0.2 M) IscS, 1.5 g (1.6 M)
MnmA, 0.38 g (1.6 M) TusA, 2.5 g (1.6 M) TusBCD, 0.49 g (1.6 M) TusE, and
0.2 g (0.4 M) tRNA (25,000 cpm). Proteins comprising the sulfur relay system, IscS,
TusA, TusBCD, and TusE, were prepared as described previously14. The reaction was
incubated at 37 ˚C for 90 min. An aliquot (6 l) was taken from the mixture, and the
tRNA was recovered by adding 30 μl ISOGEN and 6 l chloroform. The presence of
sulfur
in
the
tRNA
was
analyzed
by
gel
retardation
assay
using
[(N-acryloylamino)phenyl]mercuric chloride (APM) gel as described previously15,16. The
gel was exposed to an imaging plate, followed by analysis using a FLA7000 bioimaging
analyzer.
In vitro sulfur transfer experiment
The sulfur transfer experiment was performed as reported previously14 with slight
modifications. Ten microliters of reaction mixture consisting of 50 mM Tris-HCl (pH 7.5),
50 mM KCl, 12 mM Mg(Oac)2, 20 mM PLP, 2mM ATP, 0.8mM DTT, 0.1mM
L-[35S]cysteine (900 MBq/mmol), and 1 g of various combinations of recombinant
proteins was incubated at 37 ˚C for 10 min. The reaction mixture was mixed with sample
loading solution for SDS-PAGE to give a final concentration of 4% glycerol, 25 mM
Tris-HCl (pH 6.8), 1% SDS, and 0.0015% bromophenol blue, and incubated at 60 ˚C for
10 minutes. The reaction product was resolved by SDS-PAGE on a 15% polyacrylamide
gradient gel (Daiichi pure chemicals, Japan) for Tus proteins and tRNA and 9%
polyacrylamide gel for MnmA and IscS. The gels were dried in vacuo and exposed on an
imaging plate. [35S]-labeled sulfur was visualized by an imaging analyzer (FLA-7000,
Fujifilm).
Supplementary Information Figure Legends:
Supplementary Figure S1: The schematic of the sophisticated mechanism of precise
incorporation of a reactive sulfur into a biomolecule (tRNA), which was elucidated by the
present study. The initial tRNA binding state adopts an open conformation, which allows
both of the tRNA anticodon and solvent molecules to approach the enzyme catalytic site,
preventing the precise sulfur transfer. In the pre-reaction state, dynamic structural
rearrangements occur to form a reaction chamber (closed conformation), excluding the
solvent molecules and relocating the tRNA anticodon in close to the catalytic site.
Subsequently, the target anticodon is activated in the adenylated intermediate state. Then,
a sulfur is transferred from catalytic Cys to the target anticodon base in the closed
chamber. Finally, the gate opens and the sulfurated tRNA is released.
Supplementary Figure S2: MAD phased experimental electron densities of the Form II
crystals with the refined structure. The initial electron density was improved by density
modification with the program SOLOMON. a, Stereo view of the experimental electron
density map contoured at 1.4  around the tRNAGlu anticodon stem. b, Stereo view of the
experimental electron density map contoured at 1.0  for the minor groove interaction by
the Gly-rich loop. The carbon atoms of MnmA and tRNA are coloured cyan and pink,
respectively.
Supplementary Figure S3: Composite simulated annealing omit maps for the active
site. a, Stereoview of the composite omit map contoured at 1.0  around U34 in the Form
I crystals. b, Stereoview of the composite omit map contoured at 1.0  around the second
anticodon nucleotide U35 in the Form I crystals. c, Stereoview of the composite omit map
contoured at 0.8  for U34 in the Form II crystals. The mean real-space R-factors for the
active site are 0.225 and 0.232 in the Form I and II structures, respectively. The carbon
atoms of MnmA and tRNA are coloured cyan and yellow, respectively.
Supplementary Figure S4: Amino acid sequence alignments of MnmA family enzymes
from various organisms. Multiple alignment of each sequence was performed with
ClustalW (http://align.genome.jp). Amino acid residue numbering is based on E. coli
MnmA. The secondary structures in the pre-reaction form and adenylate intermediate
form are presented above the alignment, with the coloring scheme as follows: the
N-terminal catalytic domain is shown in blue, the variable segment is magenta, the
central domain is cyan, and C-terminal -barrel domain is red. Identical amino acids and
conservative replacements are colored in dark and light, respectively. Conserved acidic,
natural, basic, and hydrophobic residues are colored in red, green, blue, and yellow,
respectively. The P-loop motif is underlined. The residues shown with asterisks were
investigated by mutational analysis: the mutations resulting in complete loss of activity
are marked with red asterisks, those resulting in decreases in enzyme activity less than
50% of the wild-type MnmA are in green, and those resulting in reduction of the enzyme
activity to 50-80% of the wild-type MnmA are in blue. Abbreviations are as follows:
E.coli, Escherichia coli; H.infl, Haemophilus influenzae; B.subt, Bacillus subtilis; S.aure,
Staphylococcus aureus; T.mari, Thermotoga maritime; T.ther, Thermus thermophilus;
A.aeol, Aquifex aeolicus; H.sapi, Homo sapiens; M.musc, Mus musculus; D.mela,
Drosophila
melanogaster;
C.eleg,
Caenorhabditis
elegans;
S.pomb,
Schizosaccharomyces pombe; S.cere, Saccharomyces cerevisiae.
Supplementary Figure S5: Schematic representation of tRNA recognition by MnmA in
the Form I structure, highlighting the mutant analysis. Amino acid residues that
specifically interact with tRNA are mutated and shown in red. The tRNA bases in the
D-stem, anticodon stem, and anticodon loop are colored green, yellow, and blue,
respectively. The inset shows the schematic representation of the interaction between
adenylated U34 and MnmA active site in the Form III structure. Blue and red arrows
show hydrogen-bonding interactions between MnmA and tRNA mediated by protein
side-chain and protein main-chain, respectively. The dotted lines represent stacking and
van der Waals interactions.
Supplementary Figure S6: Simulated annealing omit maps contoured at 3.0  and 2.3 
for the “variable segment” of the Form I (a) and the Form II (b and c) crystals,
respectively, highlighting the structural rearrangement. The tRNA anticodon arm is
shown as a yellow ribbon. The “variable segment” is shown as a pink ball-and-stick
model, and the residual region of MnmA is shown as a dark blue stick model. Close-up
view of the omit map for the protruding -hairpin (v) in the Form II structure is
presented in panel c. In Form I (a), the “open” structure is predominantly stabilized by
hydrophobic interactions of Hv with H3a and H3b (Fig. 3a), as well as the hydrogen
bonds between Glu203 and Arg207 in Hv and the 2’-OH group of U35 (Fig. 2c). In
transition from Form I to Form II, the structural rearrangement is caused by a
reorganization of the hydrophobic interactions of the “variable segment” with H3a and
H3b (Fig. 3a, b).
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tRNAs:
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