Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis

The FemXWv aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l-Ala from Ala-tRNAAla to UDP-MurNAc-pentadepsipeptide. FemXWv is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemXWv is highly specific for incorporation of l-Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro-transcribed tRNAs indicated that the specificity of FemXWv depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G1–C72 and G2–C71 base pairs of the acceptor stem as critical for FemXWv activity in agreement with modeling of tRNAAla in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNAAla harboring nucleotide substitutions in the G3–U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemXWv. The different modes of recognition of the acceptor stem indicate that specific inhibition of FemXWv could be achieved by targeting the distal portion of tRNAAla for the design of substrate analogues

Traceless Staudinger Ligation To Introduce Chemical Diversity on β-Lactamase Inhibitors of Second Generation

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Enzymatic synthesis of vancomycin-modified DNA

International audienceMany potent antibiotics fail to treat bacterial infections due to emergence of drug-resistant strains. This surge of antimicrobial resistance (AMR) calls in for the development of alternative strategies and methods for the development of drugs with restored bactericidal activities. In this context, we surmised that identifying aptamers using nucleotides connected to antibiotics will lead to chemically modified aptameric species capable of restoring the original binding activity of the drugs and hence produce active antibiotic species that could be used to combat AMR. Here, we report the synthesis of a modified nucleoside triphosphate equipped with a vancomycin moiety on the nucleobase. We demonstrate that this nucleotide analogue is suitable for polymerase-mediated synthesis of modified DNA and importantly, highlight its compatibility with the SELEX methodology. These results pave the way for bacterial-SELEX for the identification of vancomycin-modified aptamers

Synthesis of Triazole-Linked SAM-Adenosine Conjugates: Functionalization of Adenosine at N-1 or N-6 Position without Protecting Groups

More than 150 RNA chemical modifications have been identified to date. Among them, methylation of adenosine at the N-6 position (m6A) is crucial for RNA metabolism, stability and other important biological events. In particular, this is the most abundant mark found in mRNA in mammalian cells. The presence of a methyl group at the N-1 position of adenosine (m1A) is mostly found in ncRNA and mRNA and is mainly responsible for stability and translation fidelity. These modifications are installed by m6A and m1A RNA methyltransferases (RNA MTases), respectively. In human, deregulation of m6A RNA MTases activity is associated with many diseases including cancer. To date, the molecular mechanism involved in the methyl transfer, in particular substrate recognition, remains unclear. We report the synthesis of new SAM-adenosine conjugates containing a triazole linker branched at the N-1 or N-6 position of adenosine. Our methodology does not require protecting groups for the functionalization of adenosine at these two positions. The molecules described here were designed as potential bisubstrate analogues for m6A and m1A RNA MTases that could be further employed for structural studies. This is the first report of compounds mimicking the transition state of the methylation reaction catalyzed by m1A RNA MTases

Poster Session with Peer Grading

Scientific article reading is an important competence for undergraduate students in sciences. To help acquire this skill we chose to propose students a poster session with peer evaluation. Students are grouped in teams to which subjects are assigned. Each team prepares a poster during the semester and prints it before the exam. Students then enter a rotation system in which they alternatively present their poster to their peers and one teacher, or evaluate the posters of other groups. Each session is composed of a presentation, questions and evaluation. The evaluation is performed both by the teachers and the peers. Students are also evaluated for their ability to evaluate their peers, comparing their evaluations to those performed by others (teachers and/or peers). The evaluation is normalised thanks to a dedicated evaluation grid. After 2 years of manual management, the need to scale up our approach to larger cohorts of students lead to the development of a digital system. This article details our motives and method/protocol. Finally observations, limits and improvements are reported.</p

DIAZABICYCLOOCTANE FUNCTIONALIZATION FOR INHIBITION OF -LACTAMASES FROM ENTEROBACTERIA

International audienceSecond-generation-lactamase inhibitors containing a dia abic clooctane (DBO) scaffold restore the activit of-lactams against pathogenic bacteria, including those producing class A, C, and D en mes that are not susceptible to first-generation inhibitors containing a-lactam ring. Here, e report optimi ation of a s nthetic route to access tria ole-containing DBOs and biological evaluation of a series of 17 compounds for inhibition of five-lactamases representative of en mes found in pathogenic Gram-negative bacteria. A strong correlation (Spearman coefficient of 0.87; = 4.7 10-21) as observed bet een the inhibition efficac of purified-lactamases and the potentiation of-lactam antibacterial activit indicating that DBO functionali ation did not impair penetration. In comparison to reference DBOs, avibactam and relebactam, our compounds displa ed reduced efficac likel due to the absence of h drogen bonding ith a conserved asparagine residue at position 132. This as partiall compensated b additional interactions involving certain tria ole substituents

Poster Session with Peer Grading

<p>Scientific article reading is an important competence for undergraduate students in sciences. To help acquire this skill we chose to propose students a poster session with peer evaluation. </p><p>Students are grouped in teams to which subjects are assigned. Each team prepares a poster during the semester and prints it before the exam. Students then enter a rotation system in which they alternatively present their poster to their peers and one teacher, or evaluate the posters of other groups. Each session is composed of a presentation, questions and evaluation. The evaluation is performed both by the teachers and the peers. Students are also evaluated for their ability to evaluate their peers, comparing their evaluations to those performed by others (teachers and/or peers). The evaluation is normalised thanks to a dedicated evaluation grid. </p><p>After 2 years of manual management, the need to scale up our approach to larger cohorts of students lead to the development of a digital system. This article details our motives and method/protocol. Finally observations, limits and improvements are reported.</p

Synthesis of RNA-cofactor conjugates and structural exploration of RNA recognition by an m6A RNA methyltransferase

International audienceAbstract Chemical synthesis of RNA conjugates has opened new strategies to study enzymatic mechanisms in RNA biology. To gain insights into poorly understood RNA nucleotide methylation processes, we developed a new method to synthesize RNA-conjugates for the study of RNA recognition and methyl-transfer mechanisms of SAM-dependent m6A RNA methyltransferases. These RNA conjugates contain a SAM cofactor analogue connected at the N6-atom of an adenosine within dinucleotides, a trinucleotide or a 13mer RNA. Our chemical route is chemo- and regio-selective and allows flexible modification of the RNA length and sequence. These compounds were used in crystallization assays with RlmJ, a bacterial m6A rRNA methyltransferase. Two crystal structures of RlmJ in complex with RNA–SAM conjugates were solved and revealed the RNA-specific recognition elements used by RlmJ to clamp the RNA substrate in its active site. From these structures, a model of a trinucleotide bound in the RlmJ active site could be built and validated by methyltransferase assays on RlmJ mutants. The methyl transfer by RlmJ could also be deduced. This study therefore shows that RNA-cofactor conjugates are potent molecular tools to explore the active site of RNA modification enzymes

Bisubstrate analogues as structural tools to investigate m 6 A methyltransferase active sites

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Partition of tRNAGly isoacceptors between protein and cell-wall peptidoglycan synthesis in Staphylococcus aureus

International audienceThe sequence of tRNAs is submitted to evolutionary constraints imposed by their multiple interactions with aminoacyl-tRNA synthetases, translation elongation factor Tu in complex with GTP (EF-Tu•GTP), and the ribosome, each being essential for accurate and effective decoding of messenger RNAs. In Staphylococcus aureus, an additional constraint is imposed by the participation of tRNAGly isoacceptors in the addition of a pentaglycine side chain to cell-wall peptidoglycan precursors by transferases FmhB, FemA and FemB. Three tRNAGly isoacceptors poorly interacting with EF-Tu•GTP and the ribosome were previously identified. Here, we show that these 'non-proteogenic' tRNAs are preferentially recognized by FmhB based on kinetic analyses and on synthesis of stable aminoacyl-tRNA analogues acting as inhibitors. Synthesis of chimeric tRNAs and of helices mimicking the tRNA acceptor arms revealed that this discrimination involves identity determinants exclusively present in the D and T stems and loops of non-proteogenic tRNAs, which belong to an evolutionary lineage only present in the staphylococci. EF-Tu•GTP competitively inhibited FmhB by sequestration of 'proteogenic' aminoacyl-tRNAs in vitro. Together, these results indicate that competition for the Gly-tRNAGly pool is restricted by both limited recognition of non-proteogenic tRNAs by EF-Tu•GTP and limited recognition of proteogenic tRNAs by FmhB