Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

5A, NW12A/2004G153

Structure of a complex of Pyrococcus horikoshii arginyl-tRNA synthetase andArg
tRNA with large variation of codon usage for different species
1Michiko Konno*1, Tomomi SUMIDA1, Yukie Mori1 & Ryouko Takasaka1
Ochanomizu University, Department of Chemistry, 2-1-1 Otsuka, Bunkyo-ku,Tokyo 112-8610 Japan

Introduction
Aminoacyl-tRNA synthetases catalyze aminoacylation
reaction from amino acid, tRNA and ATP to aminoacyl2+tRNA and AMP in the presence of Mg ion. Comparisonof codon usage among archaebacteria Pyrococcus
horikoshii (Ph), prokaryote E. coli (Ec), Thermus
thermophilus (Tt) and eukaryote yeast reveals that codonsof Arg, Ile and Gly have large variation for these species.
The main codon usages of Arg are CGX (X stands for
U/C/A/G) for Ec, CGG/AGG for Tt and CGX/AGA/AGG
for yeast, while AGA/AGG for Ph are 98 %. We havedetermined a crystal structure of a complex of Ph arginylArg
tRNA synthetase (ArgRS) and tRNA (CCU) andcompared this structure with crystal structures of yeast
ArgRS and Tt ArgRS and in order to clarify common
binding region of three bases of anticodon compared withMetRS, IleRS and ValRS of class Ia, the anticodon
binding domains of which have the same helix-bundle
structure as that of ArgRS.
Experiments and Results
Experiments
E. coli BL21(DE3) was transformed with a plasmid of
pET28c/ArgRS constructed from genome of Ph. Thecrude protein was purified on Ni-NTA superflow column
and with an AKTA purifier FPLC system throughcolumns Resource Q HitrapHeparin and hydroxyapatite.
The template DNA for tRNA was prepared in a large scalewith PCR by using the plasmid pUC119/ tRNAArg(CCU).
In vitro transcription of the template DNA was carried out
with T7 RNA polymerase and the crude transcript waspurified. Crystals of a complex of Ph ArgRS and tRNAArg(CCU) and a complex containing AMP-PNP wereobtained by hanging drop vapor diffusion method.
Intensity data were collected at 100 K on NW12 beamline and processed by HKL2000 and SCALEPACK. Thestructure was solved by molecular replacement methodwith MOLREP by using Tt ArgRS (PDB:1IQ0) as asearch model. The structure of tRNA (ICG) binding yeast
ArgRS (PDB:1F7V) was used as the model. Refinementsof this structure gave R factor of ca. 40%. The low
electron density maps in N-terminal domain and tRNArevealed large deviation of this domain of Ph ArgRS fromTt and yeast ArgRSs and large confor mational change ofPh tRNA from yeast tRNA. The repeats of the modelbuilding and refinements using O and CNS gave finally
Rfactor = 0.215 (Rfree = 0.259) at 2.0 Å resolution for theternary complex and Rfactor = 0.206 (Rfree = 0.266) at 2.3 Åfor the binary complex.

Result and Discussion
The ribbon model of a complex of Ph ArgRS and tRNAis shown in Fig. 1. The biochemical experiments ofmutants of MetRS and its crystal structure [1] reveal thatthree bases of the anticodon bind on the surface of helix I
and helix III and the loop connecting helix III-IV (III-IVloop). It was reported that the first base of anticodon
interacts with III-IV loop from the mutant experiments of
its loop of IleRS and MetRS. The crystal structures ofeach cognate tRNA binding on MetRS from Auifexaeolicusy [2], IleRS from S. aureus [3] and Tt ValRS [4],respectively reveal that the base of the second anticodon
Ade35 of three tRNAs resides in close proximity ofconserved Asn on helix I. On the other hands, in thecomplex of Ph ArgRS and tRNA, the base of the second
anticodon C35 form two hydrogen bonds with CO andNH of main chains of III-IV loop and overlaps in parallelto the aromatic ring of Tyr587 on helix III (Fig. 2). Threebases of anticodon for tRNA is far away from Tyr509
conserved among all investigated ArgRSs, which locatesthe position corresponding to that of conserved Asn on
helix I of MetRS, IleRS and ValRS. ArgRS has additional
N-terminal domain and the base of Ade20 of D loop oftRNA interacts with Tyr85 and Asn87 in N-terminal
domain (Fig. 1). In the crystals, this interaction may
deviate tRNA from the initial binding location.

Fig.2 anticodon binding
domain and tRNA
Fig.1 Ribbon model of Ph ArgRS and tRNA
References[1] I. Sugiura et al. Structure 8, 197 (2000).
[2] K. Nakanishi et al., Nat. Struct. Mol. Biol. 10, 931(2005).
[3] L.F. Silvian et al., Science. 285, 1074 (1999).
[4] S. Fukai et al., Cell 103, 793 (2000).
* konno@cc.ocha.ac.jp

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