tmRNA and small protein B (SmpB) are essential trans-translation system components. In the present study, we determined the crystal structure of SmpB in complex with the entire tRNA domain of the tmRNA from Thermus thermophilus. Overall, the ribonucleoprotein complex (tRNP) mimics a long-variable-arm tRNA (class II tRNA) in the canonical L-shaped tertiary structure. The tmRNA terminus corresponds to the acceptor and T arms, or the upper part, of tRNA. On the other hand, the SmpB protein simulates the lower part, the anticodon and D stems, of tRNA. Intriguingly, several amino acid residues collaborate with tmRNA bases to reproduce the canonical tRNA core layers. The linker helix of tmRNA had been considered to correspond to the anticodon stem, but the complex structure unambiguously shows that it corresponds to the tRNA variable arm. The tmRNA linker helix, as well as the long variable arm of class II tRNA, may occupy the gap between the large and small ribosomal subunits. This suggested how the tRNA domain is connected to the mRNA domain entering the mRNA channel. A loop of SmpB in the tRNP is likely to participate in the interaction with alanyl-tRNA synthetase, which may be the mechanism for the promotion of tmRNA alanylation by the SmpB protein. Therefore, the tRNP may simulate a tRNA, both structurally and functionally, with respect to aminoacylation and ribosome entry.crystal structure ͉ small protein B ͉ tmRNA ͉ trans-translation T rans-translation is an important quality control process in bacterial cells that recycles ribosomes accidentally stalled by defective mRNAs (1, 2). This system is ubiquitous in Bacteria, and is facilitated by tmRNA. Alanyl-tmRNA is delivered to the empty A site of the ribosome. Translation then resumes, using the mRNA portion of tmRNA, which encodes a tag targeted by a specific protease. Small protein B (SmpB), another key molecule for transtranslation (3), is highly conserved among all bacteria and some organelle genomes [supporting information (SI) Fig. 6]. The multifunctional roles of SmpB include alanylation enhancement of tmRNA and association with tmRNA entering the empty A site of the ribosome (4-6). The ␤-barrel structure of SmpB, revealed from two bacterial species, seems to have adapted to interact with the tmRNA to facilitate their association with translational components (7,8). The structure of the T arm and a portion of the D loop domain of tmRNA in complex with SmpB was reported, using the Aquifex aeolicus sequence (9), which revealed that the surface of the SmpB ␤-barrel structure strongly bound to the single-stranded D loop. To clarify how the tmRNA interacts with SmpB and to determine the functional mechanism on the ribosome, we solved the crystal structure of the entire tRNA domain with SmpB from Thermus thermophilus HB8. Results and DiscussionStructure Determination. To create a stable, but still functional, tRNA domain of tmRNA ( Fig. 1 A and B), several stem mutants, for slipless folding in vitro, were tested for the activation of alanylation in the presence...

Wye bases are tricyclic bases that are found in archaeal and eukaryotic tRNAs. The most modified wye base, wybutosine, which appears at position 37 (the 3'-adjacent position to the anticodon), is known to be important for translational reading-frame maintenance. Saccharomyces cerevisiae TYW1 catalyzes the tri-ring-formation step in wye-base biosynthesis, with the substrate tRNA bearing N(1)-methylated G37. Here, the crystal structure of the archaeal TYW1 homologue from Pyrococcus horikoshii is reported at 2.2 A resolution. The amino-acid sequence of P. horikoshii TYW1 suggested that it is a radical-AdoMet enzyme and the tertiary structure of P. horikoshii TYW1 indeed shares the modified TIM-barrel structure found in other radical-AdoMet enzymes. Radical-AdoMet enzymes generally contain one or two iron-sulfur (FeS) clusters. The tertiary structure of P. horikoshii TYW1 revealed two FeS cluster sites, each containing three cysteine residues. One FeS cluster site was expected from the amino-acid sequence and the other involves cysteine residues that are dispersed throughout the sequence. The existence of two FeS clusters was confirmed from the anomalous Fourier electron-density map. By superposing the P. horikoshii TYW1 tertiary structure on those of other radical-AdoMet enzymes, the AdoMet molecule, which is necessary for the reactions of radical-AdoMet enzymes, was modelled in P. horikoshii TYW1. Surface plots of conservation rates and electrostatic potentials revealed the highly conserved and positively charged active-site hollow. On the basis of the surface properties, a docking model of P. horikoshii TYW1, the tRNA, the FeS clusters and the AdoMet molecule was constructed, with the nucleoside at position 37 of tRNA flipped out from the canonical tRNA structure.

Background: Escherichia coli encodes a bifunctional oxidase/methyltransferase catalyzing the final steps of methylaminomethyluridine (mnm5U) formation in tRNA wobble positions.Results: Aquifex aeolicus encodes only a monofunctional aminomethyluridine-dependent methyltransferase, lacking the oxidase domain.Conclusion: An alternative pathway exists for mnm5U biogenesis.Significance: Information about how an organism modifies the wobble base of its tRNA is important for understanding the emergence of the genetic code.

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