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Noda Institute for Scientiﬁc Research GRANT
2011 Young Investigator Research Grant

The mechanism for biogenesis of the modiﬁed nucleosidefound in archaeal tRNAIle
Tomoyuki NUMATA
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST)

Research aims
In most archaea, the wobble cytidine (C34) of the AUAcodon speciﬁc isoleucine tRNA (tRNAIle2) is modiﬁed to 2agmatinylcytidine (agm2C), an agmatine-conjugated cytidine derivative1) (Fig. 1a). The precursor form of tRNAIle2
with unmodiﬁed C34 deciphers the methionine AUG
codon, since tRNAIle2 bears the CAU anticodon sequencethat also appears in tRNAMet. In contrast, tRNAIle2 with theagm2C modiﬁcation precisely deciphers the isoleucine
AUA codon, instead of AUG. Thus, the agm2C modiﬁcation switches the codon speciﬁcity from AUG to AUA. Theformation of agm2C is catalyzed by tRNAIle-agm2C synthetase (TiaS), using ATP and agmatine as substrates1). Although TiaS catalyzes agm2C formation in an ATP-dependent manner, a clear ATP-binding motif is not apparent inthe primary structure. Therefore, the mechanism for agm2Cformation has remained obscure. In this study, to elucidate
the mechanisms of the ATP-dependent agm2C formation
by TiaS, we determined the crystal structures of theArchaeoglobus fulgidus TiaS–tRNAIle2 complex2,3).
Methods
The recombinant A. fulgidus TiaS was produced inE. coli strain C41(DE3), and then puriﬁed. A. fulgidus

Figure 1.

tRNAIle2 was transcribed in vitro using T7 RNA polymerase, and then puriﬁed. We prepared co-crystals of theTiaS-tRNAIle2-ATP ternary complex and TiaS-tRNAIle2AMPcPP-agmatine quaternary complex. The X-ray diffraction data were collected at the beam-line BL-17A of Photon
Factory. We ﬁrst determined the TiaS-tRNAIle2-ATP ternarycomplex structure by the single-wavelength anomalous
dispersion method, at a resolution of 2.9 Å. Subsequently,
the structure of the TiaS-tRNAIle2-AMPcPP-agmatine
quaternary complex was determined at 3.1 Å resolution,by molecular replacement method.
Results
The TiaS-tRNAIle2-ATP ternary complex structure revealed that TiaS is folded into four structural domains,
comprising the TCK domain, FL domain, OB domain, andZR domain (Fig. 2). ATP is bound to the TCK domain. Thebiochemical study showed that TiaS ﬁrst hydrolyzes theATP a -b phosphate bond, and then phosphorylates C34
with the g -phosphate4) (Fig. 1b). The invariant Asp8, Asp9,and Asp11 residues are located in the vicinity of the triphosphate. The D8A, D9A, and D11A mutations abolishedthe ATP-hydrolysis and agmatine-incorporating activities.
Since ATP hydrolysis is requisite for subsequent C34 phosphorylation by TiaS, these results strongly suggest that theChemical structure and reaction scheme for the formation of 2-agmatinylcytidine (agm2C)

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TCK domain functions as the catalytic module to phosphorylate C34.
In addition to the ternary complex, we also determined
the TiaS-tRNAIle2-AMPcPP-agmatine quaternary complex
structure. A comparison between the ternary and quaternary complexes revealed structural differences, especiallyin the tRNA anticodon region (Fig. 3). In the ternary complex, C34 is located at the pocket in the FL domain, inwhich the ATP g -phosphate is too far away from C34(10 Å) for the phosphorylation reaction to occur. In contrast, C34 in the quaternary complex is kept out of thispocket (because of the presence of agmatine in this pocket),and is situated near the g -phosphate of AMPcPP (3.1 Å).
Therefore, it is suggested that the quaternary complex
adopts a productive conformation, relative to that of the ter-nary complex, with respect to the phosphorylation reaction.
Conclusion
Based on (i) a structural comparison between the ternaryand quaternary complexes, (ii) the estimated cellular concentrations of tRNAIle2 and agmatine, and (iii) the TiaS Km
values for agmatine and tRNAIle2, it is suggested that TiaS
have already bound agmatine prior to the interaction withtRNAIle2, and after tRNA binding, C34 could be directly
recognized near the ATP g -phosphate by an induced-ﬁtmechanism. In contrast, if the cellular concentration of agmatine is insufﬁcient for the modiﬁcation reaction, thenC34 could be trapped in the pocket of the FL domain, faraway from the ATP-binding site. Since TiaS has an intrinsic
basal ATP-hydrolysis activity4), regardless of the presenceor absence of the tRNA, segregation of C34 from the ATPg -phosphate is requisite to prevent the accumulation of aphosphorylated C34 intermediate (pC34). In this case, thesubsequent agmatine supply to the enzyme causes the structural transition, especially of the tRNA anticodon region, torelocate C34 adjacent to the ATP g -phosphate for the phosphorylation. In either case, the presence of agmatine inTiaS is important for properly locating C34 to the active
site, for activation by phosphorylation. Once C34 is phosphorylated, the anticodon region might become restructured
to place pC34 in the vicinity of agmatine, so the agmatine
amino group can perform the nucleophilic attack on the C2
carbon of C34, to form agm2C. The present study suggeststhat the ternary and quaternary complex structures represent the C34-trapping and pre-phosphorylation states, respectively.
References
1)

Figure 2.

Crystal structure of the TiaS-tRNAIle2-ATP ternary complex

Figure 3.

Ikeuchi, Y. et al. (2010) Agmatine-conjugated cytidine in a tRNA
anticodon is essential for AUA decoding in archaea. Nat. Chem.
Biol. 6: 277–282.
2) Osawa, T., Inanaga, H., Kimura, S., Terasaka, N., Suzuki, T., andNumata, T. (2011) Crystallization and preliminary X-ray diffraction
analysis of an archaeal tRNA-modiﬁcation enzyme, TiaS, complexedwith tRNAIle2 and ATP. Acta Crystallogr. Sect. F 67: 1414–1416.
3) Osawa, T., Kimura, S., Terasaka, N., Inanaga, H., Suzuki, T., andNumata, T. (2011) Structural basis of tRNA agmatinylation essentialfor AUA codon decoding. Nat. Struct. Mol. Biol. 18: 1275–1280.
4) Terasaka, N., Kimura, S., Osawa, T., Numata, T., and Suzuki, T.
(2011) Biogenesis of 2-agmatinylcytidine catalyzed by the dual protein and RNA kinase TiaS. Nat. Struct. Mol. Biol. 18: 1268–1274.

Structural comparison of the active sites in the ternary (a) and quaternary (b) complexes