Tsunemi Hasegawa

A hepatitis C virus (HCV) internal ribosome entry site (IRES) domain III–IV-targeted aptamer inhibits translation by binding to an apical loop of domain IIId

The hepatitis C virus (HCV) has a positive single-stranded RNA genome, and translation starts within the internal ribosome entry site (IRES) in a cap-independent manner. The IRES is well conserved among HCV subtypes and has a unique structure consisting of four domains. We used an in vitro selection procedure to isolate RNA aptamers capable of binding to the IRES domains III–IV. The aptamers that were obtained shared the consensus sequence ACCCA, which is complementary to the apical loop of domain IIId that is known to be a critical region of IRES-dependent translation. This convergence suggests that domain IIId is preferentially selected in an RNA–RNA interaction. Mutation analysis showed that the aptamer binding was sequence and structure dependent. One of the aptamers inhibited translation both in vitro and in vivo. Our results indicate that domain IIId is a suitable target site for HCV blockage and that rationally designed RNA aptamers have great potential as anti-HCV drugs.

The Role of anticodon bases and the discriminator nucleotide in the recognition of some E. coli tRNAs by their aminoacyl-tRNA synthetases

SummaryThe T7 polymerase transcription system was used for in vitro synthesis of unmodified versions of the E. coli tRNA mutants that insert asparagine, cysteine, glycine, histidine, and serine. These tRNAs were used to qualitatively explore the role of some anticodon bases and the discriminator nucleotide in the recognition of tRNA by aminoacyl-tRNA synthetases. Coupled with data from earlier studies, these new results essentially complete a survey of all E. coli tRNAs with respect to the involvement of anticodon bases and the discriminator nucleotide in tRNA recognition. It is found that in the vast majority of tRNAs both of these elements are significant components of tRNA identity. This is not universally true, however. Anticodon sequences are unimportant in tRNAser, tRNALeu, and tRNAAla while the discriminator base is inconsequential in tRNAser and tRNAThr. The significance of these results for origin-of-life studies is discussed.

Identity determinants of E. coli threonine tRNA

To investigate the identity determinants of E. coli threonine tRNA, various transcripts were prepared by in vitro transcription system with T7 RNA polymerase. Substitutions of the anticodon second letter G35 and the third letter U36 to other nucleotides led to a remarkable decrease of threonine charging activity. Charging experiments with a series of anticodon-deletion transcripts also suggest the importance of the G35U36 sequence. A mutation at either the G1-C72 or C2-G71 base pair in the acceptor stem seriously affected the threonine charging activity. These results indicate that the second and third positions of the anticodon and the first and second base pairs in the acceptor stem are the recognition sites of E. coli tRNA(THR) for threonyl-tRNA synthetase. Discriminator base, A73, is not involved in threonine charging activity.

Increased inhibitory ability of conjugated RNA aptamers against the HCV IRES

Hepatitis C virus (HCV) translation begins within the internal ribosome entry site (IRES). We have previously isolated two RNA aptamers, 2-02 and 3-07, which specifically bind to domain II and domain III-IV of the HCV IRES, respectively, and inhibit IRES-dependent translation. To improve the function of these aptamers, we constructed two conjugated molecules of 2-02 and 3-07. These bound to the target RNA more efficiently than the two parental aptamers. Furthermore, they inhibited IRES-dependent translation about 10 times as efficiently as the 3-07 aptamer. This result indicates that combining aptamers for different target recognition sites potentiates the inhibition activity by enhancing the domain-binding efficiency.

Modulation of Double-stranded RNA Recognition by the N-terminal Histidine-rich Region of the Human Toll-like Receptor 3

Toll-like receptors (TLRs) are an essential component of the innate immune response to microbial pathogens. TLR3 is localized in intracellular compartments, such as endosomes, and initiates signals in response to virus-derived double-stranded RNA (dsRNA). The TLR3 ectodomain (ECD), which is implicated in dsRNA recognition, is a horseshoe-shaped solenoid composed of 23 leucine-rich repeats (LRRs). Recent mutagenesis studies on the TLR3 ECD revealed that TLR3 activation depends on a single binding site on the nonglycosylated surface in the C-terminal region, comprising H539 and several asparagines within LRR17 to -20. TLR3 localization within endosomes is required for ligand recognition, suggesting that acidic pH is the driving force for TLR3 ligand binding. To elucidate the pH-dependent binding mechanism of TLR3 at the structural level, we focused on three highly conserved histidine residues clustered at the N-terminal region of the TLR3 ECD: His39 in the N-cap region, His60 in LRR1, and His108 in LRR3. Mutagenesis of these residues showed that His39, His60, and His108 were essential for ligand-dependent TLR3 activation in a cell-based assay. Furthermore, dsRNA binding to recombinant TLR3 ECD depended strongly on pH and dsRNA length and was reduced by mutation of His39, His60, and His108, demonstrating that TLR3 signaling is initiated from the endosome through a pH-dependent binding mechanism, and that a second dsRNA binding site exists in the N-terminal region of the TLR3 ECD characteristic solenoid. We propose a novel model for the formation of TLR3 ECD dimers complexed with dsRNA, which incorporates this second binding site.

Identity determinants of E. coli tRNAVal

In order to study the identity elements of valine tRNA, various transcripts of E. coli valine tRNA mutants were constructed. Both mutations at the second letter A35 of the anticodon and at the discriminator base A73 seriously damaged valine charging activity. Mutations at either the G3-C70 or U4-A69 base pairs in the acceptor stem also affected the activity. Only one nucleotide substitution of the second letter G35 of the anticodon with A35 brought an 18% valine charging activity into alanine tRNA, which acquired an almost full charging activity with valine by introducing an additional change at those two base pairs in the acceptor stem. These results indicate that the second letter A35 of the anticodon, discriminator base and acceptor stem are involved in recognition by valyl-tRNA synthetase.

Novel sugar-binding specificity of the type XIII xylan-binding domain of a family F/10 xylanase from Streptomyces olivaceoviridis E-86.

The type XIII xylan‐binding domain (XBD) of a family F/10 xylanase (FXYN) from Streptomyces olivaceoviridis E‐86 was found to be structurally similar to the ricin B chain which recognizes the non‐reducing end of galactose and specifically binds to galactose containing sugars. The crystal structure of XBD [Fujimoto, Z. et al. (2000) J. Mol. Biol. 300, 575–585] indicated that the whole structure of XBD is very similar to the ricin B chain and the amino acids which form the galactose‐binding sites are highly conserved between the XBD and the ricin B chain. However, our investigation of the binding abilities of wt FXYN and its truncated mutants towards xylan demonstrated that the XBD bound xylose‐based polysaccharides. Moreover, it was found that the sugar‐binding unit of the XBD was a trimer, which was demonstrated in a releasing assay using sugar ranging in size from xylose to xyloheptaose. These results indicated that the binding specificity of the XBD was different from those of the same family lectins such as the ricin B chain. Somewhat surprisingly, it was found that lactose could release the XBD from insoluble xylan to a level half of that observed for xylobiose, indicating that the XBD also possessed the same galactose recognition site as the ricin B chain. It appears that the sugar‐binding pocket of the XBD has evolved from the ancient ricin super family lectins to bind additional sugar targets, resulting in the differences observed in the sugar‐binding specificities between the lectin group (containing the ricin B chain) and the enzyme group.

Sugar-complex structures of the C-half domain of the galactose-binding lectin EW29 from the earthworm Lumbricus terrestris

R-type lectins are one of the most prominent types of lectin; they exist ubiquitously in nature and mainly bind to the galactose unit of sugar chains. The galactose-binding lectin EW29 from the earthworm Lumbricus terrestris belongs to the R-type lectin family as represented by the plant lectin ricin. It shows haemagglutination activity and is composed of a single peptide chain that includes two homologous domains: N-terminal and C-terminal domains. A truncated mutant of EW29 comprising the C-terminal domain (rC-half) has haemagglutination activity by itself. In order to clarify how rC-half recognizes ligands and shows haemagglutination activity, X-ray crystal structures of rC-half in complex with D-lactose and N-acetyl-D-galactosamine have been determined. The structure of rC-half is similar to that of the ricin B chain and consists of a beta-trefoil fold; the fold is further divided into three similar subdomains referred to as subdomains alpha, beta and gamma, which are gathered around the pseudo-threefold axis. The structures of sugar complexes demonstrated that subdomains alpha and gamma of rC-half bind terminal galactosyl and N-acetylgalactosaminyl glycans. The sugar-binding properties are common to both ligands in both subdomains and are quite similar to those of ricin B chain-lactose complexes. These results indicate that the C-terminal domain of EW29 uses these two galactose-binding sites for its function as a single-domain-type haemagglutinin.

Leucyl/Phenylalanyl-tRNA-Protein Transferase-Mediated Chemoenzymatic Coupling of N-Terminal Arg/Lys Units in Post-translationally Processed Proteins with Non-natural Amino Acids

Position-specific incorporation of non-natural amino acids through ribosomal systems can introduce functional groups into proteins at desired positions. Through the use of multiple four-base codons, we have successfully incorporated two or even three non-natural amino acids into single proteins in vitro. Because at least five mutually orthogonal four-base codons have been identified, up to five non-natural amino acids can, in theory, be incorporated into single proteins, but the limited decoding efficiency of the four-base codons means that triple mutation is the current limit. Further incorporation of non-natural amino acids into specific positions of proteins needs another technique. We and other groups have introduced a strategy for the biosynthetic incorporation of a probe only at the N terminus of a nascent protein. At this stage, an E. coli aminoacyl-tRNA synthetase mixture was used to charge initiator methionine tRNA with methionine, and the a-amino group of the MettRNA was then treated with an amine-reactive chemical probe. In this way the probe could be incorporated at the Nterminal ends of several proteins, but the approach is currently limited by low incorporation efficiency and by the production of only limited quantities of the labeled protein in a batch process. Enzymatic modification of proteins without the participation of ribosomes is an alternative approach to the position-specific introduction of functional groups. Leucyl/phenylalanyl ACHTUNGTRENNUNG(L/F)tRNA-protein transferase from E. coli is known to catalyze the transfer of hydrophobic amino acids—such as phenylalanine, leucine, or methionine—from tRNA to the N termini of proteins possessing lysine or arginine as their N-terminal residues. N-terminal arginine and lysine are secondary destabilizing residues in E. coli because the destabilization depends on their conjugation to the leucine or phenylalanine primary destabilizing residues by the transferase. Here we report that this enzymatic coupling can be expanded to link non-natural amino acids to the N termini of target proteins through the use of tRNAs aminoacylated with various types of non-natural amino acid. Because this new technique can be used independently of non-natural amino acid mutagenesis, it provides another method through which to add a single extra non-natural amino acid, in addition to the incorporation of multiple non-natural amino acids through orthogonal four-base codons. Proteins expressed in bacteria normally contain methionine units at their N termini, except in cases in which the next amino acid is a small one, so we developed a novel and convenient procedure to attach single Lys units to the N termini of proteins, choosing SoCBM13—known as a xylan-binding domain (XBD)—as our target protein. We cloned the SoCBM13 coding sequence into a pET27 plasmid vector to obtain an expressing plasmid pET27-Lys-SoCBM13. The vector contains a pelB signal peptide coding region directly before the cloning site (see Supporting Information). By introducing this plasmid into E. coli we expressed a fusion protein of pelB signal peptide and Lys-SoCBM13. After translation, the pelB signal peptide is spontaneously cleaved off in E. coli, leaving a single Lys unit and providing the desired Lys-SoCBM13. As a negative control, we also prepared His-SoCBM13, in which a single histidine unit is attached to the N terminus. Successful syntheses of the extended SoCBM13 were confirmed by TOF-MS, as ACHTUNGTRENNUNGdescribed in the Supporting Information. Initially we chose 3-nitrotyrosine as the non-natural amino acid, as it can easily be detected by use of an anti-nitrotyrosine antibody in Western blotting. E. coli tRNA was aminoacylated with nitrotyrosine with use of a resin-supported ribozyme for aminoacylation and the resulting ntrTyr-tRNA was added to a reaction mixture containing the extended SoCBM13 and the transferase. The products were analyzed by Western blotting and visualized with the anti-nitrotyrosine antibody (Figure 1). A band containing the ntrTyr unit was detected only in the presence of Lys-SoCBM13 and ntrTyr-tRNA (lane 1), while no ntrTyr-linked protein was detected in the mixture of HisSoCBM13 and ntrTyr-tRNA (lane 2), or in the mixture of LysSoCBM13 and free tRNA (lane 3). These results clearly indicate that the non-natural amino acid had successfully been coupled to Lys-SoCBM13. The presence and the position of the ntrTyr unit were examined by a sequence analysis of the product based on Edman [a] Prof. Dr. M. Taki, S. Matoba, Y. Kobayashi, Prof. Dr. J. Futami, Prof. Dr. M. Sisido Department of Bioscience and Biotechnology Faculty of Engineering, Okayama University 3-1-1 Tsushimanaka, Okayama 700-8530 (Japan) Fax: (+81)86-251-8219 E-mail : taki@biotech.okayama-u.ac.jp sisido@cc.okayama-u.ac.jp [b] Prof. Dr. M. Taki, Dr. A. Kuno, Prof. Dr. K. Taira National Institute of Advanced Industrial Science and Technology (AIST) 1-1-1 Higashi, Tsukuba Science City, 305-8562 (Japan) [c] Prof. Dr. H. Murakami, Prof. Dr. H. Suga, Prof. Dr. K. Taira Department of Chemistry and Biotechnology Graduate School of Engineering, Tokyo University Hongo, Tokyo 113-8656 (Japan) [d] Prof. Dr. T. Hasegawa Department of Material and Biological Chemistry Faculty of Science, Yamagata University Yamagata 990-8560 (Japan) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Identity elements of Thermus thermophilus tRNAThr

In this study, we identified nucleotides that specify aminoacylation of tRNAThr by Thermus thermophilus threonyl‐tRNA synthetase (ThrRS) using in vitro transcripts. Mutation studies showed that the first base pair in the acceptor stem as well as the second and third positions of the anticodon are major identity elements of T. thermophilus tRNAThr, which are essentially the same as those of Escherichia coli tRNAThr. The discriminator base, U73, also contributed to the specific aminoacylation, but not the second base pair in the acceptor stem. These findings are in contrast to E. coli tRNAThr, where the second base pair is required for threonylation, with the discriminator base, A73, playing no roles. In addition, among several mutations at the third base pair in the acceptor stem, only the G3‐U70 mutant was a poor substrate for ThrRS, suggesting that the G3‐U70 wobble pair, which is the identity determinant of tRNAAla, acts as a negative element for ThrRS. Similar results were obtained in E. coli and yeast. Thus, this manner of rejection of tRNAAla is also likely to have been retained in the threonine system throughout evolution.