Hiroki Kanazawa

Abstract Post-transcriptional modifications modulate transfer RNA (tRNA) structure, stability, and codon decoding properties, contributing to translation regulation and adaptation across diverse organisms, including bacterial pathogens. We provide a comprehensive analysis of tRNA modifications in Staphylococcus aureus using extensive oligonucleotide mass spectrometry and deep-sequencing methods, generating a high-confidence modification map for each individual tRNA species, including non-proteogenic tRNAGly. While the overall tRNA modification landscape is conserved among Gram-positive bacteria, our data uncovered unexpected S. aureus-specific features. These include the absence of m2A37 in tRNAs despite the presence of the methyltransferase RlmN, a single multi-site DusB2 enzyme catalyzing all tRNA dihydrouridylation, and evidence suggesting a dedicated pseudouridine synthase responsible for Ψ32. Besides, heterogeneous modification patterns were observed in tRNALeu(UAA) and tRNALys(UUU), highlighting a complex interplay in anticodon hypermodification. Time-course proteomics revealed dynamic expression of tRNA modifying enzymes during growth. Integration of ribosome profiling and Nanopore tRNA sequencing offered a global view of S. aureus decoding properties, revealing efficient four-way wobble recognition, slower translation of rare codons by low abundant tRNAs, and distinctive decoding dynamics of Gly codons potentially influenced by the unusual modification status of tRNAGly(UCC). This work establishes a framework to dissect the role of tRNA modifications in S. aureus physiology and pathogenesis.

A single strand version of DNA ₂ -[Ag ₁₆ Cl ₂ ] ⁸⁺ was created by bridging the two DNA strands with a thymine segment.

Abstract Aminoglycosides (AG) are antibiotics that lower the accuracy of protein synthesis by targeting a highly conserved RNA helix of the ribosomal A‐site. The discovery of AGs that selectively target the eukaryotic ribosome, but lack activity in prokaryotes, are promising as antiprotozoals for the treatment of neglected tropical diseases, and as therapies to read‐through point‐mutation genetic diseases. However, a single nucleobase change A1408G in the eukaryotic A‐site leads to negligible affinity for most AGs. Herein we report the synthesis of 6′‐fluorosisomicin, the first 6′‐fluorinated aminoglycoside, which specifically interacts with the protozoal cytoplasmic rRNA A‐site, but not the bacterial A‐site, as evidenced by X‐ray co‐crystal structures. The respective dispositions of 6′‐fluorosisomicin within the bacterial and protozoal A‐sites reveal that the fluorine atom acts only as a hydrogen‐bond acceptor to favorably interact with G1408 of the protozoal A‐site. Unlike aminoglycosides containing a 6′‐ammonium group, 6′‐fluorosisomicin cannot participate in the hydrogen‐bonding pattern that characterizes stable pseudo‐base‐pairs with A1408 of the bacterial A‐sites. Based on these structural observations it may be possible to shift the biological activity of aminoglycosides to act preferentially as antiprotozoal agents. These findings expand the repertoire of small molecules targeting the eukaryotic ribosome and demonstrate the usefulness of fluorine as a design element.