Download RNA Helicase Module in an Acetyltransferase That Modifies a

Chimnaronk et al.
Supplemental Data
RNA Helicase Module in an Acetyltransferase That
Modifies a Specific tRNA Anticodon
Sarin Chimnaronk, Tateki Suzuki, Tetsuhiro Manita, Yoshiho Ikeuchi, Min Yao, Tsutomu Suzuki, and
Isao Tanaka
Supplemental Discussion
Implications for Eukaryotic Homologs of TmcA in rRNA Maturation
It is interesting that the same enzymatic module is used for acetylation of both RNA and protein such as
histone. Could an ancestral acetylase have acted on RNA in the primordial RNA World? If so, there should
be traces reminiscent of such a molecule in either eukaryotes or archaea. Indeed, BLAST analysis with E.
coli TmcA as a query identified homologous genes containing consecutive DUF699 and Acetyltranf_1 motifs
in all eukaryotes and archaea (except for methanogens and Thermoplasma) (Figure S3). The modified
nucleoside ac4C at the wobble position is found only in some γ-proteobacterial and haloarchaeal tRNAs
(Sprinzl & Vassilenko, 2005). Intriguingly, in halophiles, ac4C naturally occurs in five tRNA species for
Met
glutamine, glutamate, lysine, proline, and serine, bearing C34, but not in the elongator tRNA
. A definite
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role of ac C at the wobble position in halophiles has not been elucidated but may be different from bacterial.
Besides the wobble position, ac4C has been found only at position 12 in the D stems of tRNA
tRNA
Ser
and
Leu
, both of which have extra-long variable arms, in eukaryotes (Rafalski et al, 1979). However, an
earlier report identified a dissimilar nonessential gene, TAN1, responsible for ac4C formation at position 12 in
Ser
CGA
Saccharomyces cerevisiae tRNA
(Johansson & Bystrom, 2004), and thus, the eukaryotic homologs of
TmcA are not likely involved in modifying tRNA.
Clues to the biological function of the TmcA homologs unexpectedly emerged from nucleoside analysis
of ribosomal RNA, revealing that the small subunit (ssu) rRNA contains ac4C conserved in both archaea and
eukaryotes from yeast to mammals, but not in bacteria (Noon et al, 1998; Thomas et al, 1978). In S.
cerevisiae, a protein homologous to TmcA is KRE33 (the open reading frame YNL132w) whose heterozygous
mutant exhibits haploinsufficiency in K1 killer toxin resistance (Page et al, 2003). Remarkably, KRE33 is an
essential protein accumulating in the nucleolus (Huh et al, 2003). In addition, the recent proteomic analysis
of protein complexes from yeast illustrated the interactions of KRE33 with several ribosomal proteins and a
subset of ribosomal processing factors (Figure S6) (Gavin et al, 2006; Grandi et al, 2002). These facts shed
light on the as yet unclear functions of the TmcA homologs as being involved in rRNA modification and
maturation in eukaryotic organisms. Furthermore, an earlier fascinating work detected an ac4C present in the
short terminal helix at the 3´-end of 18S ssu rRNA from Dictyostelium discoideum (McCarroll et al, 1983).
This terminal helix is preserved among eukaryotic species and also bears sequences complementary to the
evolutionally conserved U13 snoRNA that acts as a trans-acting factor in faithful nucleolytic cleavage at the
3΄-end of 18S rRNA (Figure S6) (Cavaille et al, 1996). These pose an attractive question as to the hidden
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relationship between RNA acetylation and the processing of 18S rRNA in eukaryotes. Why are the
eukaryotic TmcA homologs essential for cell viability? Does U13 snoRNA guide TmcA homolog to a
modified site in rRNA? Is the formation of ac4C in 18S rRNA required for faithful processing at the 3′-end?
Our structural and biochemical studies may place us at the beginning of unraveling the mystery of rRNA
maturation in eukaryotic nuclei.
Supplemental Methods
Mass Spectrometry
An LCQDUO ion-trap (IT) mass spectrometer (ThermoFinnigan) equipped with an electrospray ionization
(ESI) source and HP1100 liquid chromatography system (Agilent Technologies) was used to analyze the
nucleosides. An aliquot (20 µg) of the total RNA was digested with P1 nuclease (3 µg, Yamasa, Japan) and
alkaline phosphatase (0.04 units, from E. coli C75, Takara, Japan) in a 25 μL reaction mixture containing of
20 mM Hepes-KOH (pH 7.6) at 37 °C for 3 hours. The hydrolysate was fractionated using an Inertsil ODS-3
column, 250 × 2.1 mm (GL science, Japan). The solvent system consisted of 5 mM NH4OAc (pH 5.3) (A)
and 60% acetonitrile (B), used as follows: 1-35% B in 0-35min., 35-99% B in 35-40min., 99% B in
40-50min. The chromatographic effluent (150 μL/mL) was directly conducted into the ion source without
prior splitting. Positive ions were scanned over an m/z range of 103 to 700 throughout the separation under
the following conditions: flow rate of sheath gas, 95 arb; capillary temperature, 245°C; spray voltage, 5 kV.
Crystallization, Data Collection and Structure Determination
For initial crystallization screening, the hanging-drop vapour-diffusion method was used with commercially
available screening kits (Hampton Research and Emerald BioStructures). Each drop was prepared by mixing
1 μl of protein solution (5 mg/ml) with an equal volume of reservoir solution and was vapor-equilibrated
against 250 μl of reservoir solution at 18ºC. Thin-plate crystals diffracting x-ray to approximately 8 Å were
obtained with a reservoir solution containing 50 mM tri-sodium citrate (pH 5.5), 15% (w/v) PEG 5000
monomethyl ether, and 50 mM lithium sulfate within a few days only when acetyl-CoA was included in the
protein drop. Over a couple of weeks, well-shaped orthorhombic crystals with dimensions of 0.2 mm × 0.2
mm × 0.05 mm fortuitously appeared in the same drop; however, reproducing these crystals required the
streak-seeding technique (Figure S2). Optimal Se-Met-labeled TmcA crystals were grown in the presence of
1 mM ATP, 1 mM acetyl-CoA, and 2 mM MgCl2. The crystals were cryo-protected with reservoir solution
supplemented with 25% (v/v) glycerol, and then two data sets were successfully collected at beamline BL6A
and ARNW12 stations of Photon Factory (Tsukuba, Japan) under cryogenic condition (100 K). These were
indexed, integrated, scaled, and merged using the HKL2000 package (Otwinowski & Minor, 1997). Crystals
exhibited the symmetry of space group P212121, and contained two TmcA molecules per asymmetric unit.
The best crystal diffracted to a minimum Bragg spacing (dmin) of about 2.3 Å.
The phases were obtained by the multi wavelength anomalous diffraction (MAD) method at 2.9 Å
resolution with 19 of 22 Se sites identified by using the SOLVE program (Terwilliger & Berendzen, 1999).
Subsequent phase improvements with the RESOLVE program (Terwilliger, 2000) followed by DM (Cowtan
& Main, 1996) in the CCP4i suite (Consortium, 1994) yielded an electron density map of satisfactory clarity
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by which an initial model building was constructed using RESOLVE (Figure S2). Subsequently, the resulting
peptide fragments were manually composed with the graphics program O (Jones et al, 1991), and complete
model building was automatically achieved and refined with the program LAFIRE (Yao et al, 2006; Zhou et
al, 2006) running together with CNS (Brunger et al, 1998). Afterward, high-resolution data were obtained
and the phase problem was solved by the molecular replacement method with CNS (Brunger et al, 1998). At
this stage, models of acetyl-CoA and adenine nucleoside were built using TURBO (Roussel & Cambillau,
1991) according to the Fo-Fc difference electron density map. Iterative rounds of manual refinement with
Coot (Emsley & Cowtan, 2004) followed by automatic refinement by LAFIRE yielded the final model with a
crystallographic R factor of 23.4% and an Rfree factor of 27.4%. The refined structure of TmcA was validated
by the programs PROCHECK (Laskowski et al, 1993) and WHATIF (Vriend, 1990). The summary of data
statistics is presented in Table 1. Secondary structure elements of proteins were assigned with the program
WHATIF (Vriend, 1990), and all molecular graphics were created using PyMol (DeLano, 2002).
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Supplemental Figures
Figure S1. Chemical Structure of the Modified Ribonucleosides that Govern Decoding Rules for the
CAU Anticodon of tRNA
Cloverleaf representations of two tRNA species from E. coli bearing the same CAU anticodons with different
modifications at the wobble cytidine bases. tRNAMet, responsible for decoding the AUG codon has
N4-acetylcytidine (ac4C), whereas tRNAIle, corresponding to AUA decoding, has lysidine (k2C). Bases
involved in specific recognition by TmcA and TilS, according to earlier tRNA mutagenesis studies (Ikeuchi
et al, 2008; Ikeuchi et al, 2005), are illustrated in red and blue characters, respectively.
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Figure S2. Co-Crystallization of E. coli TmcA with Acetyl-CoA and ATP, and Electron Density Map
Quality
(A) Thin-plate crystals of recombinant E. coli TmcA were initially obtained in 66% Crystal Screen II
condition No. 26 (66 mM MES pH 6.5, 20% PEG MME 5000, and 132 mM ammonium sulfate,
Hampton Research). Optimizing around this condition leaded to a different crystal form grown in the
same drop (left panel), which diffracted to higher resolution. The Se-Met-labeled TmcA crystals (right
panel) were only obtained by the cross streak-seeding technique with a crushed native crystal.
(B) The FOM-weight experimental electron density map (2.76 Å, contour level 2.0 σ), calculated after
solvent flattening by DM (Cowtan & Main, 1996), in a top-down view of monomer A as in Figure 2B
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(see text) is drawn in green (top left). The map is superimposed on the refined model. For comparison,
the σ-weighted |2Fo-Fc| (purpleblue) and |Fo-Fc| (red) difference electron density maps calculated by
LAFIRE (Yao et al, 2006; Zhou et al, 2006) in a late state of refinement at 2.35 Å, contoured at 2.0 σ and
5.0 σ, respectively, are also shown in the same orientation for monomer A (top right) and B (bottom left).
Note that the ADP-binding site in monomer A is occupied by a sulfate ion in monomer B (indicated by
white arrows). The bottom right panel shows the different electron density map in the vicinity of the
acetyl-CoA binding site in monomer A.
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Figure S3. Sequence Analysis of TmcA Homologs from Three Domains of Life
(A) The amino-acid sequences of TmcA homologs from bacterial (green), archaeal (blue), and eukaryotic
(magenta) sources found by BLASTP search engine were aligned using ClustalW (Thompson et al,
1994). The phylogenetic tree was drawn using TreeView (Page, 1996). Scale bar represents 0.1 amino
acid replacements per site.
(B) Sequence alignments of TmcA homologs are shown together with the secondary structure elements as
defined by the crystal structure. Solvent accessibility (acc) is shown in blue, cyan and white for
accessible, intermediate and buried residues, respectively. Conserved amino acids are in white in
redfilled rectangles. Similar residues are in red, surrounded by blue lines. Characteristic motifs of TmcA
are indicated by boxes colored as in Figure 3B. Residues involved in ADP binding are indicated under
the sequence with open and filled triangles for protein main chain and side chain, respectively. Residues
involved in acetyl-CoA recognition are also demonstrated with ellipses in the same manner. Point
mutations examined in this study are indicated by black stars above the sequence. Alignment was
composed using ESPript (Gouet et al, 1999).
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Figure S4. Architecture of the Acetyl-CoA Binding Site and Proposed Reaction Mechanism
(A) Superposition of Cα traces of E. coli TmcA (body domain) with the GNAT fold from other protein
acetyltransferases. TmcA is colored in yellow, whereas the GCN5 histone acetyltransferase complexed
with a CoA from Tetrahymena thermophilus (1PU9, Clements et al, 2003) and Rimi, ribosomal S18
N-alpha acetyltransferase complexed with an acetyl-CoA from Salamonella Typhimurium (2CNS) are in
purple and cyan, respectively. A bound acetyl-CoA to TmcA is shown as stick representation. The right
panel shows the conformational comparison among their ligands in which the pyrophosphate groups are
superimposed.
(B) Schematic diagram of acetyl-CoA recognition site in TmcA. Protein is in light gray and hydrogen-bond
interactions are indicated as blue dashed line labeled with bond lengths. An acetyl moiety is emphasized
in red, and a black dashed line indicates the distance from the nearest atom to the carbonyl oxygen of
acetyl group. Hydrophobic interactions (orange) were analyzed by the LIGPLOT program (Wallace et al,
1995).
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Figure S5. Representative Mutational Analyses Data
(A) Mass spectrometric (LC/MS) analysis of the total nucleosides obtained from the E. coli ΔtmcA strain
supplied with wild-type or mutant TmcA plasmids. For the wild-type, the UV trace at 254 nm (top panel),
mass chromatograms detecting lysidine (m/z 372, middle panel), and ac4C (m/z 286, bottom panel) are
shown. Only mass chromatograms at m/z 286 for detecting ac4C are shown for mutants. Peaks
corresponding to ac4C are indicated by red arrows.
(B) Thin layer chromatography (TLC) ATP hydrolysis assay with the recombinant wild-type and mutant
proteins (see experimental procedures for detail).
(C) Electrophoretic mobility shift assay (EMSA) with purified TmcA mutants. Constant protein amount (50
Met
pmol) was examined with increasing tRNA
(0-200 pmol).
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Figure S6. Implication for the Eukaryotic TmcA Homologs in Ribosomal RNA Maturation
(A) Protein-protein interaction network of yeast TmcA homolog (KRE33) obtained from the BioGRID
database (http://thebiogrid.org, Stark et al, 2006). Only proteins with a score of socio-affinity index over
5.0 (http://yeastcomplexes.embl.de, Gavin et al, 2006) are selected in order to represent real direct
contacts. Proteins are colored according to their functions (orange, ribosomal protein; brown, protein
kinase; green, ribosomal RNA processing). The line length reflects socio-affinity indices (higher scores
are shorter).
(B) Secondary structure of the 3´-terminal helix of human 18S rRNA reveals the complementary sequences
with a functionally uncharacterized U13 snoRNA. Modifications found in this region are colored in dark
gray (Piekna-Przybylska et al, 2008), and ac4C is highlighted in green (McCarroll et al, 1983).
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