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...

Fragile X syndrome is caused by loss of FMR1 protein expression. FMR1 binds RNA and associates with polysomes in the cytoplasm; thus, it has been proposed to function as a regulator of gene expression at the posttranscriptional level. Posttranslational modification of FMR1 had previously been suggested to regulate its activity, but no experimental support for this model has been reported to date. Here we report that FMR1 in Drosophila melanogaster (dFMR1) is phosphorylated in vivo and that the homomer formation and the RNAbinding activities of dFMR1 are modulated by phosphorylation in vitro. Identification of a protein phosphorylating dFMR1 showed it to be Drosophila casein kinase II (dCKII). dCKII directly interacts with and phosphorylates dFMR1 in vitro. The phosphorylation site in dFMR1 was identified as Ser406, which is highly conserved among FMR1 family members from several species. Using mass spectrometry, we established that Ser406 of dFMR1 is indeed phosphorylated in vivo. Furthermore, human FMR1 (hFMR1) is also phosphorylated in vivo, and alteration of the conserved Ser500 in hFMR1 abolishes phosphorylation by CKII in vitro. These studies support the model that the biological functions of FMR1, such as regulation of gene expression, are likely regulated by its phosphorylation.Fragile X syndrome is the most frequent cause of inheritable mental retardation and is also one of the most common singlegene disorders (14). Cognitive deficits reported for fragile X children range from mild to severe, and behavioral disturbances include social and attention deficits, autistic-like behaviors, unusual responses to sensory stimuli, hyperactivity, and sleep problems (12,14). The gene directly responsible for fragile X syndrome, FMR1, was identified in 1991 (47). In most cases, the syndrome is caused by a trinucleotide repeat expansion in the 5Ј untranslated region of the FMR1 gene (49). The expansion of the CGG repeat results in an absence of the encoded protein. It is therefore clear that the pathophysiological mechanisms leading to symptoms in fragile X syndrome can be elucidated by studying the functions of the FMR1 gene.The fact that the FMR1 protein is a cytoplasmic protein with RNA-binding motifs (two KH domains and an RGG box) (2, 38, 39) and the fact that it associates with ribosomes (6, 9, 21, 41) suggest that this protein functions in the posttranscriptional regulation of some specific mRNA expression (16). Indeed, FMR1 has recently been proposed to be a translational repressor in a cell-free system and in frog oocytes (26,27). Interestingly, although the inhibitory effect was demonstrated not to be influenced by the features of the mRNAs used in those studies, FMR1 seemed to specifically inhibit its own translation (36). It has been known that FMR1 is found at postsynaptic sites enriched in ribosomes, where some specific mRNAs, including FMR1, are translated in response to synaptic activation (13). It has also been observed that in fragile X patients the numbers of dendritic spines are different and the mo...

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The hypermodified nucleoside N(6)-threonylcarbamoyladenosine resides at position 37 of tRNA molecules bearing U at position 36 and maintains translational fidelity in the three kingdoms of life. The N(6)-threonylcarbamoyl moiety is composed of L-threonine and bicarbonate, and its synthesis was genetically shown to require YrdC/Sua5. YrdC/Sua5 binds to tRNA and ATP. In this study, we analyzed the L-threonine-binding mode of Sua5 from the archaeon Sulfolobus tokodaii. Isothermal titration calorimetry measurements revealed that S. tokodaii Sua5 binds L-threonine more strongly than L-serine and glycine. The Kd values of Sua5 for L-threonine and L-serine are 9.3 μM and 2.6 mM, respectively. We determined the crystal structure of S. tokodaii Sua5, complexed with AMPPNP and L-threonine, at 1.8 Å resolution. The L-threonine is bound next to AMPPNP in the same pocket of the N-terminal domain. Thr118 and two water molecules form hydrogen bonds with AMPPNP in a unique manner for adenine-specific recognition. The carboxyl group and the side-chain hydroxyl and methyl groups of L-threonine are buried deep in the pocket, whereas the amino group faces AMPPNP. The L-threonine is located in a suitable position to react together with ATP for the synthesis of N(6)-threonylcarbamoyladenosine.

Various molecular phylogenetic analyses suggest that group I introns in fungi and terrestrial ⁄ nonaquatic plants were horizontally transmitted multiple times in the course of evolution among distantly related species [1][2][3]. We have shown this is also the case for algal mitochondrial introns [4,5] Group I introns are thought to be self-propagating mobile elements, and are distributed over a wide range of organisms through horizontal transmission. Intron invasion is initiated through cleavage of a target DNA by a homing endonuclease encoded in an open reading frame (ORF) found within the intron. The intron is likely of no benefit to the host cell and is not maintained over time, leading to the accumulation of mutations after intron invasion. Therefore, regular invasional transmission of the intron to a new species at least once before its degeneration is likely essential for its evolutionary long-term existence. In many cases, the target is in a proteincoding region which is well conserved among organisms, but contains ambiguity at the third nucleotide position of the codon. Consequently, the homing endonuclease might be adapted to overcome sequence polymorphisms at the target site. To address whether codon degeneracy affects horizontal transmission, we investigated the recognition properties of a homing enzyme, I-CsmI, that is encoded in the intronic ORF of a group I intron located in the mitochondrial COB gene of the unicellular green alga Chlamydomonas smithii. We successfully expressed and purified three types of N-terminally truncated I-CsmI polypeptides, and assayed the efficiency of cleavage for 81 substrates containing single nucleotide substitutions. We found a slight but significant tendency that I-CsmI cleaves substrates containing a silent or tolerated amino acid change more efficiently than nonsilent or nontolerated ones. The published recognition properties of I-SpomI, I-ScaI, and I-SceII were reconsidered from this point of view, and we detected proficient adaptation of I-SpomI, I-ScaI, and I-SceII for target site sequence degeneracy. Based on the results described above, we propose that intronic homing enzymes are adapted to cleave sequences that might appear at the target region in various species, however, such adaptation becomes less prominent in proportion to the time elapsed after intron invasion into a new host.