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The stringent response is critical for the survival of Mycobacterium tuberculosis (Mtb) under nutrient starvation. The mechanism is mediated by a GTP pyrophosphokinase known as Rel, containing N-terminal synthetase and hydrolase domains and C-terminal regulatory domains, which include the TGS domain (ThrRS, GTPase, and SpoT proteins) that has been proposed to activate the synthetase domain via interaction with deacylated tRNA. Here, we present the NMR solution structure of the Mtb Rel TGS domain (MtRel TGS), consisting of five antiparallel β-strands and one helix-loop-helix motif. The interaction of MtRel TGS with deacylated tRNA is shown, indicating the critical amino acids of MtRel TGS in tRNA binding, and presenting the first structural evidence of MtRel TGS binding to deacylated tRNA in solution in the absence of the translational machinery. Ministry of Education (MOE) We would like to thank the Singapore Ministry of Education Academic Research Fund Tier 1 (Rg137/15)for the funding to GG.

Staphylococcus aureus RsaG is a 3′-untranslated region (3′UTR) derived sRNA from the conserved uhpT gene encoding a glucose-6-phosphate (G6P) transporter expressed in response to extracellular G6P. The transcript uhpT-RsaG undergoes degradation from 5′- to 3′-end by the action of the exoribonucleases J1/J2, which are blocked by a stable hairpin structure at the 5′-end of RsaG, leading to its accumulation. RsaG together with uhpT is induced when bacteria are internalized into host cells or in the presence of mucus-secreting cells. Using MS2-affinity purification coupled with RNA sequencing, several RNAs were identified as targets including mRNAs encoding the transcriptional factors Rex, CcpA, SarA, and the sRNA RsaI. Our data suggested that RsaG contributes to the control of redox homeostasis and adjusts metabolism to changing environmental conditions. RsaG uses different molecular mechanisms to stabilize, degrade, or repress the translation of its mRNA targets. Although RsaG is conserved only in closely related species, the uhpT 3′UTR of the ape pathogen S. simiae harbors an sRNA, whose sequence is highly different, and which does not respond to G6P levels. Our results hypothesized that the 3′UTRs from UhpT transporter encoding mRNAs could have rapidly evolved to enable adaptation to host niches., This work was supported by the Centre National de la Recherche Scientifique (CNRS), by the French National Research Agency ANR (ANR-18-CE12-0025-04 CoNoCo to P.R.). This work of the Interdisciplinary Thematic Institute IMCBio, as part of the ITI 2021–2028 program of the University of Strasbourg, CNRS, and Inserm was supported by IdEx Unistra (ANR-10-IDEX-0002), SFRI-STRAT'US (ANR 20-SFRI-0012), and by EUR IMCBio (IMCBio ANR-17-EURE-0023) under the framework of the French Investments for the Future Program. ED and LB were supported by the “Fondation pour la Recherche Médicale” (FDT201904007957 and ECO202006011534)

In many bacteria, the stabilities and functions of small regulatory RNAs (sRNAs) that act by base pairing with target RNAs most often are dependent on Hfq or ProQ/FinO-domain proteins, two classes of RNA chaperone proteins. However, while all bacteria appear to have sRNAs, many have neither Hfq nor ProQ/FinO-domain proteins raising the question of whether another factor might act as an sRNA chaperone in these organisms. Several recent studies have reported that KH domain proteins, such as KhpA and KhpB, bind sRNAs. Here we describe what is known about the distribution, structures, RNA-binding properties, and physiologic roles of KhpA and KhpB and discuss evidence for and against these proteins serving as sRNAs chaperones.

Background Although transfer RNA (tRNA) has been found to be the main source of a rich class of non-coding RNA. Moreover, the tRNA-derived small RNA (tsRNA) has been proved to play an irreplaceable role in the human body, and its dynamic imbalance could affect the progress of the disease. However, the research on tsRNA in oral submucous fibrosis (OSF) is still scarce. Methods We sequenced the OSF and validated it by PCR. We found that there were significant differences in their expression levels in OSF. Furthermore, bioinformatic analysis was performed to explore the roles of these fragments in oral submucous fibrosis. Results 126 tsRNAs in OSF were dysregulated, including 73 up-regulated tsRNAs and 53 down-regulated tsRNAs. The down-regulated tiRNA-Val-CAC-002, tRF-Asn-GTT-005, tRF-Trp-CCA-007 and up-regulated tRF-Gly-TCC-016, tRF-Pro-TGG-009 showed significant differences by qRT-PCR validation, which were consistent with the results of RNA sequencing. Gene Ontology and pathway analysis revealed that tRF-Gly-TCC-016 would possibly promote the formation and progress of OSF through cytokine-cytokine receptor interaction and cAMP signal pathway, while tiRNA-Val-CAC-002 could be primarily concerned with the transition from OSF to oral squamous cell carcinoma (OSCC). Conclusion tRNA-derived fragments are dysregulated and could be involved in the pathogenesis of oral submucous fibrosis. tRF-Gly-TCC-016 and tiRNA-Val-CAC-002 may be new regulatory molecules that could affect the process of OSF by regulating signal pathways through interacting with multiple genes.

Ribonuclease P (RNase P), which is required for the 5'-end maturation of tRNAs in every organism, has been shown to play a limited role in other aspects of RNA metabolism in Escherichia coli. Using RNA-sequencing (RNA-seq), we demonstrate that RNase P inactivation affects the abundances of ~46% of the expressed transcripts in E. coli and provide evidence that its essential function is its ability to generate pre-tRNAs from polycistronic tRNA transcripts. The RNA-seq results agreed with the published data and northern blot analyses of 75/83 transcripts (mRNAs, sRNAs, and tRNAs). Changes in transcript abundances in the RNase P mutant also correlated with changes in their half-lives. Inactivating the stringent response did not alter the rnpA49 phenotype. Most notably, increases in the transcript abundances were observed for all genes in the cysteine regulons, multiple toxin-antitoxin modules, and sigma S-controlled genes. Surprisingly, poly(A) polymerase (PAP I) modulated the abundances of ~10% of the transcripts affected by RNase P. A comparison of the transcriptomes of RNase P, RNase E, and RNase III mutants suggests that they affect distinct substrates. Together, our work strongly indicates that RNase P is a major player in all aspects of post-transcriptional RNA metabolism in E. coli.

Under conditions of nutrient adversity, bacteria adjust metabolism to minimise cellular energy usage. This is often achieved by controlling the synthesis and degradation of RNA. In Escherichia coli, RNase E is the central enzyme involved in RNA degradation and serves as a scaffold for the assembly of the multiprotein complex known as the RNA degradosome. The activity of RNase E against specific mRNAs can also be regulated by the action of small RNAs (sRNA). In this case, the ubiquitous bacterial chaperone Hfq bound to sRNAs can interact with the RNA degradosome for the sRNA guided degradation of target RNAs. The RNA degradosome and Hfq have never been visualised together in live bacteria. We now show that in long-term nitrogen starved E. coli, both RNase E and Hfq co-localise in a single, large focus. This subcellular assembly, which we refer to as the H-body, forms by a liquid-liquid phase separation type mechanism and includes components of the RNA degradosome, namely, the helicase RhlB and the exoribonuclease polynucleotide phosphorylase. The results support the existence of an hitherto unreported subcellular compartmentalisation of a process(s) associated with RNA management in stressed bacteria.

Background tRNA‐derived fragments (tRFs) have been found to play a regulatory role in the occurrence and development of many tumors. The aim of this study was to identify the expression of tRFs in breast cancer and their ability to serve as diagnostic markers for breast cancer. Methods Total RNA was extracted from breast cancer and paracancerous tissues (n = 83), as well as from the sera of breast cancer patients (n = 214) and healthy donors (n = 113) using trizol reagents. Expression of tRFs was then detected by q‐PCR, and analyzed using t‐test and ROC to illuminate their potential as biomarkers for breast cancer. Results Our results demonstrated that tRFs: tRF‐Gly‐CCC‐046, tRF‐Tyr‐GTA‐010 and tRF‐Pro‐TGG‐001 were downregulated in both tissues and sera from breast cancer patients as well as early‐stage patients compared with those in the healthy donors. More importantly, the three tRFs were capable of serving as circulating biomarkers of diagnostics and early diagnosis of breast cancer, possessing areas under the curve (AUC) of 0.7871 and 0.7987, respectively. Conclusions tRFs: tRF‐Gly‐CCC‐046, tRF‐Tyr‐GTA‐010 and tRF‐Pro‐TGG‐001 are downregulated in breast cancer and early breast cancer and act as new potential biomarkers for the diagnosis and early diagnosis of breast cancer.

Post-transcriptional modification of nucleosides is observed in almost all elements of RNA. Modified nucleosides finely tune the structure of RNA molecules and affect vital functions, such as the modified wobble position 34 of transfer RNAs expanding the reading preference of anticodons to codons. Recent investigations have revealed that the modification species and their frequencies in an RNA element are not stable but vary with specific cellular factors including metabolites and particular proteins (writers, readers, and erasers). To understand the link between dynamic RNA modifications and biological processes, sensitive and reliable methods for determining modified nucleosides are required. In this study, micro-flow (8 μL/min) hydrophilic interaction liquid chromatography was coupled with triple quadrupole mass spectrometry for the simultaneous determination of adenosine, uridine, cytidine, guanosine, and 20 modified nucleosides. The method was calibrated using 0.1-1000 nM standards (∼0.03-300 ng/mL) and successfully applied to the determination of transfer RNA modifications in the model cyanobacterium Synechococcus elongatus PCC 7942. A protocol for the isolation of a clean transfer RNA pool was optimized, requiring only 25 ng for the identification and quantification of transfer RNA modifications. This micro-flow liquid chromatography-tandem mass spectrometry method constitutes the first step toward monitoring dynamic ribonucleoside modifications in a limited RNA sample.

The carbon storage regulator system and base-pairing small RNAs (sRNAs) represent two predominant modes of bacterial post-transcriptional regulation, which globally influence gene expression. Binding of CsrA protein to the 5' UTR or initial mRNA coding sequences can affect translation, RNA stability, and/or transcript elongation. Base-pairing sRNAs also regulate these processes, often requiring assistance from the RNA chaperone Hfq. Transcriptomics studies in Escherichia coli have identified many new CsrA targets, including Spot 42 and other base-pairing sRNAs. Spot 42 synthesis is repressed by cAMP-CRP, induced by the presence of glucose, and Spot 42 post-transcriptionally represses operons that facilitate metabolism of nonpreferred carbon sources. CsrA activity is also increased by glucose via effects on CsrA sRNA antagonists, CsrB/C. Here, we elucidate a mechanism wherein CsrA binds to and protects Spot 42 sRNA from RNase E-mediated cleavage. This protection leads to enhanced repression of srlA by Spot 42, a gene required for sorbitol uptake. A second, independent mechanism by which CsrA represses srlA is by binding to and inhibiting translation of srlM mRNA, encoding a transcriptional activator of srlA. Our findings demonstrate a novel means of regulation, by CsrA binding to a sRNA, and indicate that such interactions can help to shape complex bacterial regulatory circuitry.

Summary RNase P functions either as a catalytic ribonucleoprotein (RNP) or as an RNA‐free polypeptide to catalyse RNA processing, primarily tRNA 5′ maturation. To the growing evidence of non‐canonical roles for RNase P RNP subunits including regulation of chromatin structure and function, we add here a role for the rice RNase P Rpp30 in innate immunity. This protein (encoded by LOC_Os11g01074) was uncovered as the top hit in yeast two‐hybrid assays performed with the rice histone deacetylase HDT701 as bait. We showed that HDT701 and OsRpp30 are localized to the rice nucleus, OsRpp30 expression increased post‐infection by Pyricularia oryzae (syn. Magnaporthe oryzae), and OsRpp30 deacetylation coincided with HDT701 overexpression in vivo. Overexpression of OsRpp30 in transgenic rice increased expression of defence genes and generation of reactive oxygen species after pathogen‐associated molecular pattern elicitor treatment, outcomes that culminated in resistance to a fungal (P. oryzae) and a bacterial (Xanthomonas oryzae pv. oryzae) pathogen. Knockout of OsRpp30 yielded the opposite phenotypes. Moreover, HA‐tagged OsRpp30 co‐purified with RNase P pre‐tRNA cleavage activity. Interestingly, OsRpp30 is conserved in grass crops, including a near‐identical C‐terminal tail that is essential for HDT701 binding and defence regulation. Overall, our results suggest that OsRpp30 plays an important role in rice immune response to pathogens and provides a new approach to generate broad‐spectrum disease‐resistant rice cultivars.

A major challenge in the field of RNA chemistry is the identification of selective and quantitative conversion reactions on RNA that can be used for tagging and any other RNA tool development. Here, we introduce metal‐free diazotransfer on native RNA containing an aliphatic primary amino group using the diazotizing reagent fluorosulfuryl azide (FSO2N3). The reaction provides the corresponding azide‐modified RNA in nearly quantitatively yields without affecting the nucleobase amino groups. The obtained azido‐RNA can then be further processed utilizing well‐established bioortho‐gonal reactions, such as azide‐alkyne cycloadditions (Click) or Staudinger ligations. We exemplify the robustness of this approach for the synthesis of peptidyl‐tRNA mimics and for the pull‐down of 3‐(3‐amino‐3‐carboxypropyl)uridine (acp3U)‐ and lysidine (k2C)‐containing tRNAs of an Escherichia coli tRNA pool isolated from cellular extracts. Our approach therefore adds a new dimension to the targeted chemical manipulation of diverse RNA species., Metal‐free diazotransfer on native RNA using the reagent FSO2N3 selectively provides azide‐modified RNA in quantitative yields. The stereochemically controlled reaction opens new avenues for the synthesis of RNA‐peptide conjugates and for the isolation of cellular RNA containing post‐transcriptional nucleotide modifications with an aliphatic primary amino group, such as acp3U.

Chloroplasts are semiautonomous organelles found in photosynthetic plants. The major functions of chloroplasts include photosynthesis and carbon fixation, which are mainly regulated by its circular genomes. In the highly conserved chloroplast genome, the chloroplast transfer RNA genes (cp tRNA) play important roles in protein translation within chloroplasts. However, the evolution of cp tRNAs remains unclear. Thus, in the present study, we investigated the evolutionary characteristics of chloroplast tRNAs in five Adoxaceae species using 185 tRNA gene sequences. In total, 37 tRNAs encoding 28 anticodons are found in the chloroplast genome in Adoxaceae species. Some consensus sequences are found within the Ψ‐stem and anticodon loop of the tRNAs. Some putative novel structures were also identified, including a new stem located in the variable region of tRNATyr in a similar manner to the anticodon stem. Furthermore, phylogenetic and evolutionary analyses indicated that synonymous tRNAs may have evolved from multiple ancestors and frequent tRNA duplications during the evolutionary process may have been primarily caused by positive selection and adaptive evolution. The transition and transversion rates are uneven among different tRNA isotypes. For all tRNAs, the transition rate is greater with a transition/transversion bias of 3.13. Phylogenetic analysis of cp tRNA suggested that the type I introns in different taxa (including eukaryote organisms and cyanobacteria) share the conserved sequences “U‐U‐x2‐C” and “U‐x‐G‐x2‐T,” thereby indicating the diverse cyanobacterial origins of organelles. This detailed study of cp tRNAs in Adoxaceae may facilitate further investigations of the evolution, phylogeny, structure, and related functions of chloroplast tRNAs., In this study, we determined the evolution of chloroplast tRNA genes of five selected Adoxaceae species. We found that synonymous tRNAs may evolved from multiple ancestors. During the evolution process, the dominated tRNA duplications were mainly caused by positive selection and adaptive evolution, while tRNA transfers and losses by various complex mechanisms.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the gold-standard technique to study RNA and its various modifications. While most research on RNA nucleosides has been focused on their biological roles, discovery of new modifications remains of interest. With state-of-the-art technology, the presence of artifacts can confound the identification of new modifications. Here, we report the characterization of a non-natural mcm5 isoC ribonucleoside in S. cerevisiae total tRNA hydrolysate by higher-energy collisional dissociation (HCD)-based fingerprints and isotope labeling of RNA. Its discovery revealed a class of amino/imino ribonucleoside artifacts that are generated during RNA hydrolysis under ammonium-buffered mild basic conditions. We then identified digestion conditions that can reduce or eliminate their formation. These finding and method enhancements will improve the accurate detection of new RNA modifications.

Transfer RNAs (tRNAs) are fundamental molecules in cellular translation. In this study we have highlighted a fluorescence-based perceptive approach for tRNAs by using a quinoxaline small molecule. We have synthesised a water-soluble fluorescent pyrimido-quinoxaline-fused heterocycle containing a mandatory piperazine tail (DS1) with a large Stokes shift (∼160 nm). The interaction between DS1 and tRNA results in significant fluorescence enhancement of the molecule with Kd ∼5 μM and multiple binding sites. Our work reveals that the DS1 binding site overlaps with the specific Mg2+ ion binding site in the D loop of tRNA. As a proof-of-concept, the molecule inhibited Pb2+ -induced cleavage of yeast tRNAPhe in the D loop. In competitive binding assays, the fluorescence of DS1-tRNA complex is quenched by a known tRNA-binder, tobramycin. This indicates the displacement of DS1 and, indeed, a substantiation of specific binding at the site of tertiary interaction in the central region of tRNA. The ability of compound DS1 to bind tRNA with a higher affinity compared to DNA and single-stranded RNA offers a promising approach to developing tRNA-based biomarker diagnostics in the future.

Following stress, transfer RNA (tRNA) is cleaved to generate tRNA halves (tiRNAs). These tiRNAs have been shown to repress protein translation. Angiogenin was considered the main enzyme that cleaves tRNA at its anticodon to generate 35-45 nucleotide long tiRNA halves, however, the recent reports indicate the presence of angiogenin-independent cleavage. We previously observed tRNA cleavage pattern occurring away from the anticodon site. To explore this noncanonical cleavage, we analyze tRNA cleavage patterns in rat model of ischemia-reperfusion and in two rat cell lines. In vivo mitochondrial tRNAs were prone to this noncanonical cleavage pattern. In vitro, however, cytosolic and mitochondrial tRNAs could be cleaved noncanonically. Our results show an important regulatory role of mitochondrial stress in angiogenin-mediated tRNA cleavage. Neither angiogenin nor RNH1 appear to regulate the noncanonical tRNA cleavage. Finally, we verified our previous findings of the role of Alkbh1 in regulating tRNA cleavage and its impact on noncanonical tRNA cleavage.

Genetic code reprogramming is a powerful approach to controlled protein modification. A remaining challenge, however, is the generation of vacant codons. We targeted the initiation machinery of E. coli, showing that restriction of the formyl donor or inhibition of the formyl transferase during in vitro translation is sufficient to prevent formylation of the acylated initiating tRNA and thereby create a vacant initiation codon that can be reprogrammed by exogenously charged tRNA. Our approach conveniently generates peptides and proteins tagged N‐terminally with non‐canonical functional groups at up to 99 % reprogramming efficiency, in combination with decoding the AUG elongation codons either with native methionine or with further reprogramming with azide‐ and alkyne‐containing cognates. We further show macrocyclization and intermolecular modifications with these click handles, thus emphasizing the applicability of our method to current challenges in peptide and protein chemistry., An approach is presented for the facile generation of a vacant initiation codon, based on preventing formylation of the endogenous initiator. This can be achieved by either removing its substrate or adding an inhibitor, and it enables the generation of peptides and proteins reprogrammed with multiple and diverse functional groups and reactive handles.

Sea cucumber species are abundant (>1400 species) and widely distributed globally. mtDNA sequencing is frequently used to identify the phylogenetic and evolutionary relationships among species. However, there are no reports on the mitochondrial genome of Phyllophorus liuwutiensis. Here, we performed mtDNA sequencing of P. liuwutiensis to examine its phylogenetic relationships with other echinoderms. Its mitochondrial genome (15 969 bp) contains 37 coding genes, including 13 protein‐coding genes, 22 tRNA genes and 2 rRNA genes. Except for one protein‐coding gene (nad6) and five tRNA genes encoded on the negative strand, all other genes were encoded on the positive strand. The mitochondrial bases of P. liuwutiensis were composed of 29.55% T, 22.16% C, 35.64% A and 12.64% G. The putative control region was 703 bp in length. Seven overlapping regions (1–10 bp) were found. The noncoding region between the genes ranged from 1 to 130 bp in length. One putative control region has been found in the P. liuwutiensis mitogenome. All of the tRNA genes were predicted to fold into a cloverleaf structure. In addition, we compared the gene arrangements of six echinoderms, revealing that the gene order of P. liuwutiensis was a new arrangement., In this paper, we analyzed the mitochondrial genome of Phyllophorus liuwutiensis. We examined genome composition, base composition, codon usage of protein‐coding genes and gene arrangement. In addition, we compared the gene arrangements of six echinoderms, revealing that the gene order of P. liuwutiensis was a new arrangement.

Genetic code expansion (GCE) is a versatile tool to site‐specifically incorporate a noncanonical amino acid (ncAA) into a protein, for example, to perform fluorescent labeling inside living cells. To this end, an orthogonal aminoacyl‐tRNA‐synthetase/tRNA (RS/tRNA) pair is used to insert the ncAA in response to an amber stop codon in the protein of interest. One of the drawbacks of this system is that, in order to achieve maximum efficiency, high levels of the orthogonal tRNA are required, and this could interfere with host cell functionality. To minimize the adverse effects on the host, we have developed an inducible GCE system that enables us to switch on tRNA or RS expression when needed. In particular, we tested different promotors in the context of the T‐REx or Tet‐On systems to control expression of the desired orthogonal tRNA and/or RS. We discuss our result with respect to the control of GCE components as well as efficiency. We found that only the T‐REx system enables simultaneous control of tRNA and RS expression., Genetic code expansion on demand by combining the amber suppression technology with the T‐REx system. Amber suppression is regulated by the T‐REx system to switch on the tRNA production and/or the synthetase translation only when needed, thus minimizing the risk of adverse effects for the cells resulting from over‐production of unused tRNA or synthetase.

Cytoplasmic aminoacyl-tRNA synthetases (ARSs) are organized into multi-tRNA synthetase complexes (MSCs), from Archaea to mammals. An evolutionary conserved role of the MSCs is enhancement of aminoacylation by forming stable associations of the ARSs and tRNAs. In mammals, a single macromolecular MSC exists, which is composed of eight cytoplasmic ARSs, for nine amino acids, and three scaffold proteins. Consequently, nearly half of aminoacyl-tRNA efflux becomes concentrated at the MSC. Stable supply of aminoacyl-tRNA to the ribosome is, therefore, considered to be a major role of the mammalian MSC. Furthermore, the mammalian MSC also serves as a reservoir for releasable components with noncanonical functions. In this study, a split-luciferase complementation system was applied to investigate the configuration of the MSC in live mammalian cells. Multiplex interconnections between the components were simplified into binary protein-protein interactions, and pairwise comparison of the interactions reconstituted a framework consistent with previous in vitro studies. Reversibility of the split-luciferase reporter binding demonstrated convertible organization of the mammalian MSC, including interferon gamma (IFNγ)-stimulated glutamyl-prolyl-tRNA synthetase 1 (EPRS1) release, as well as the cooperation with the ribosome bridged by the tRNAs. The cell-based analysis provided an improved understanding of the flexible framework of the mammalian MSC in physiological conditions.

Objective Mitochondrial diseases are a group of genetic diseases caused by mutations in mitochondrial DNA and nuclear DNA, among which, mutations in mitochondrial tRNA genes possessing prominent status. In most of the cases, however, the detailed molecular pathogenesis of these tRNA gene mutations remains unclear. Methods We performed the clinical emulation, muscle histochemistry, northern blotting analysis of tRNA levels, biochemical measurement of respiratory chain complex activities and mitochondrial respirations in muscle tissue and cybrid cells. Results We found a novel m.4349C>T mutation in mitochondrial tRNAGln gene in a patient present with encephalopathy, epilepsy, and deafness. We demonstrated molecular pathomechanisms of this mutation. This mutation firstly disturbed the translation machinery of mitochondrial tRNAGlnand impaired mitochondrial respiratory chain complex activities, followed by remarkable mitochondrial dysfunction and ROS production. Interpretation. This study illustrated the pathogenicity of a novel m.4349C>T mutation and provided a better understanding of the phenotype associated with mutations in mitochondrial tRNAGln gene.

Many bacterial small RNAs (sRNAs) efficiently inhibit translation of target mRNAs by forming a duplex that sequesters the Shine-Dalgarno (SD) sequence or start codon and prevents formation of the translation initiation complex. There are a growing number of examples of sRNA-mRNA binding interactions distant from the SD region, but how these mediate translational regulation remains unclear. Our previous work in Escherichia coli and Salmonella identified a mechanism of translational repression of manY mRNA by the sRNA SgrS through a binding interaction upstream of the manY SD. Here, we report that SgrS forms a duplex with a uridine-rich translation-enhancing element in the manY 5’ untranslated region. Notably, we show that the enhancer is ribosome-dependent and that the small ribosomal subunit protein S1 interacts with the enhancer to promote translation of manY. In collaboration with the chaperone protein Hfq, SgrS interferes with the interaction between the translation enhancer and ribosomal protein S1 to repress translation of manY mRNA. Since bacterial translation is often modulated by enhancer-like elements upstream of the SD, sRNA-mediated enhancer silencing could be a common mode of gene regulation.

Emerging evidence indicates that small RNAs, including microRNAs (miRNAs) and their isoforms (isomiRs), and transfer RNA fragments (tRFs), are differently expressed in breast cancer (BC) and can be detected in blood circulation. Circulating small RNAs and small RNAs in extracellular vesicles (EVs) have emerged as ideal markers in small RNA‐based applications for cancer detection. In this study, we first undertook small RNA sequencing to assess the expression of circulating small RNAs in the serum of BC patients and cancer‐free individuals (controls). Expression of 3 small RNAs, namely isomiR of miR‐21‐5p (3′ addition C), miR‐23a‐3p and tRF‐Lys (TTT), was significantly higher in BC samples and was validated by small RNA sequencing in an independent cohort. Our constructed model using 3 small RNAs showed high diagnostic accuracy with an area under the receiver operating characteristic curve of 0.92 and discriminated early‐stage BCs at stage 0 from control. To test the possibility that these small RNAs are released from cancer cells, we next examined EVs from the serum of BC patients and controls. Two of the 3 candidate small RNAs were identified, and shown to be abundant in EVs of BC patients. Interestingly, these 2 small RNAs are also more abundantly detected in culture media of breast cancer cell lines (MCF‐7 and MDA‐MB‐231). The same tendency in selective elevation seen in total serum, serum EV, and EV derived from cell culture media could indicate the efficiency of this model using total serum of patients. These findings indicate that small RNAs serve as significant biomarkers for BC detection., EV small RNA in breast cancer can be found in serum small RNA biomarker.

Queuosine (Q) is a hypermodified RNA nucleoside that is found in tRNAHis, tRNAAsn, tRNATyr, and tRNAAsp. It is located at the wobble position of the tRNA anticodon loop, where it can interact with U as well as C bases located at the respective position of the corresponding mRNA codons. In tRNATyr and tRNAAsp of higher eukaryotes, including humans, the Q base is for yet unknown reasons further modified by the addition of a galactose and a mannose sugar, respectively. The reason for this additional modification, and how the sugar modification is orchestrated with Q formation and insertion, is unknown. Here, we report a total synthesis of the hypermodified nucleoside galactosyl‐queuosine (galQ). The availability of the compound enabled us to study the absolute levels of the Q‐family nucleosides in six different organs of newborn and adult mice, and also in human cytosolic tRNA. Our synthesis now paves the way to a more detailed analysis of the biological function of the Q‐nucleoside family., The first total synthesis of the hypermodified RNA nucleoside galactosyl‐queuosine (galQ) was achieved. Moreover, the distribution of this nucleoside as well as mannosyl‐queuosine and queuosine was determined in tissues of newborn and adult mice, and in human cytosolic tRNAs.

Various pathogenic variants in both mitochondrial tRNAPhe and Phenylalanyl-tRNA synthetase mitochondrial protein coding gene (FARS2) gene encoding for the human mitochondrial PheRS have been identified and associated with neurological and/or muscle-related pathologies. An important Guanine-34 (G34)A anticodon mutation associated with myoclonic epilepsy with ragged red fibers (MERRF) syndrome has been reported in hmit-tRNAPhe . The majority of G34 contacts in available aaRSs-tRNAs complexes specifically use that base as an important tRNA identity element. The network of intermolecular interactions providing its specific recognition also largely conserved. However, their conservation depends also on the invariance of the residues in the anticodon binding domain (ABD) of human mitochondrial Phenylalanyl-tRNA synthetase (hmit-PheRS). A defect in recognition of the anticodon of tRNAPhe may happen not only because of G34A mutation, but also due to mutations in the ABD. Indeed, a pathogenic mutation in FARS2 has been recently reported in a 9-year-old female patient harboring a p.Asp364Gly mutation. Asp364 is hydrogen bonded (HB) to G34 in WT hmit-PheRS. Thus, there are two pathogenic variants disrupting HB between G34 and Asp364: one is associated with G34A mutation, and the other with Asp364Gly mutation. We have measured the rates of tRNAPhe aminoacylation catalyzed by WT hmit-PheRS and mutant enzymes. These data ranked the residues making a HB with G34 according to their contribution to activity and the signal transduction pathway in the hmit-PheRS-tRNAPhe complex. Furthermore, we carried out extensive MD simulations to reveal the interdomain contact topology on the dynamic trajectories of the complex, and gaining insight into the structural and dynamic integrity effects of hmit-PheRS complexed with tRNAPhe . DATABASE: Structural data are available in PDB database under the accession number(s): 3CMQ, 3TUP, 5MGH, 5MGV.

Summary Even when they no longer require the presence of iron, cells use zinc as a divalent cation, involved in a large variety of catalytic and regulatory functions. This metal is so important that it appears that ribosomes are instrumental in its ultimate storage. Here, we summarize a detailed analysis which investigates the way the global cell metabolism is integrated by zinc. This integration results from the zinc‐dependent way in which the one‐carbon metabolism is always coupled to the translation process, not only via methionine and S‐adenosylmethionine, but via the complex set‐up of the modification of the position 34 of the anticodon of tRNAs., Global metabolic integration results from the zinc‐dependent way in which the one‐carbon metabolism is coupled to the translation process, via methionine, S‐adenosylmethionine, and the complex set up of the position 34 modification of the anticodon of tRNAs.

Aminoacyl‐tRNA synthetases (AARSs) charge tRNA with their cognate amino acids. Many other enzymes use amino acids as substrates, yet discrimination against noncognate amino acids that threaten the accuracy of protein translation is a hallmark of AARSs. Comparing AARSs to these other enzymes allowed us to recognize patterns in molecular recognition and strategies used by evolution for exercising selectivity. Overall, AARSs are 2–3 orders of magnitude more selective than most other amino acid utilizing enzymes. AARSs also reveal the physicochemical limits of molecular discrimination. For example, amino acids smaller by a single methyl moiety present a discrimination ceiling of ~200, while larger ones can be discriminated by up to 105‐fold. In contrast, substrates larger by a hydroxyl group challenge AARS selectivity, due to promiscuous H‐ bonding with polar active site groups. This ‘hydroxyl paradox’ is resolved by editing. Indeed, when the physicochemical discrimination limits are reached, post‐transfer editing – hydrolysis of tRNAs charged with noncognate amino acids, evolved. The editing site often selectively recognizes the edited noncognate substrate using the very same feature that the synthetic site could not efficiently discriminate against. Finally, the comparison to other enzymes also reveals that the selectivity of AARSs is an explicitly evolved trait, showing some clear examples of how selection acted not only to optimize catalytic efficiency with the target substrate, but also to abolish activity with noncognate threat substrates (‘negative selection’).

Background Mitochondrial dysfunctions caused by mitochondrial DNA (mtDNA) pathogenic mutations play putative roles in type 2 diabetes mellitus (T2DM) progression. But the underlying mechanism remains poorly understood. Methods A large Chinese family with maternally inherited diabetes and deafness (MIDD) underwent clinical, genetic, and molecular assessment. PCR and sequence analysis are carried out to detect mtDNA variants in affected family members, in addition, phylogenetic conservation analysis, haplogroup classification, and pathogenicity scoring system are performed. Moreover, the GJB2, GJB3, GJB6, and TRMU genes mutations are screened by PCR-Sanger sequencing. Results Six of 18 matrilineal subjects manifested different clinical phenotypes of diabetes. The average age at onset of diabetic patients is 52 years. Screening for the entire mitochondrial genomes suggests the co-existence of two possibly pathogenic mutations: tRNATrp A5514G and tRNASer(AGY) C12237T, which belongs to East Asia haplogroup G2a. By molecular level, m.A5514G mutation resides at acceptor stem of tRNATrp (position 3), which is critical for steady-state level of tRNATrp . Conversely, m.C12237T mutation occurs in the variable region of tRNASer(AGY) (position 31), which creates a novel base-pairing (11A-31T). Thus, the mitochondrial dysfunctions caused by tRNATrp A5514G and tRNASer(AGY) C12237T mutations, may be associated with T2DM in this pedigree. But we do not find any functional mutations in those nuclear genes. Conclusion Our findings suggest that m.A5514G and m.C12337T mutations are associated with T2DM, screening for mt-tRNA mutations is useful for molecular diagnosis and prevention of mitochondrial diabetes.

Here, we present a multi-cycle chemoenzymatic synthesis of modified RNA with simplified solid-phase handling to overcome size limitations of RNA synthesis. It combines the advantages of classical chemical solid-phase synthesis and enzymatic synthesis methods using magnetic streptavidin beads and biotinylated RNA. Successful introduction of light-controllable RNA nucleotides into the tRNAMet sequence was confirmed by gel electrophoresis and mass spectrometry. The methods tolerate modifications in the RNA phosphodiester backbone and allow introductions of photocaged and photoswitchable nucleotides as well as photocleavable strand breaks and fluorophores.

While a number of approaches have been developed to analyze liquid chromatography tandem mass spectrometry (LC-MS/MS) data obtained from modified oligonucleotides, the majority of these methods require analyzing every MS/MS spectrum de novo to sequence the oligonucleotide and place the modification. Spectral matching is an alternative approach for analyzing MS/MS data that is based on creating a library of annotated MS/MS spectra against which individual MS/MS data can be searched. Here, we have adapted the existing NIST spectral matching software to enable its use in the interpretation of MS/MS data obtained from modified oligonucleotides. In particular, we demonstrate the utility of this approach to identify specific post-transcriptionally modified nucleosides in particular transfer RNAs (tRNAs) obtained through a conventional RNA modification mapping experimental protocol. Spectral matching was found to be an efficient approach for screening for known modified tRNAs by using the experimental data as the library and previously annotated RNase T1 digestion products of tRNAs as the reference spectra. The utility of spectral matching for rapid analysis of multiple LC-MS/MS analyses was demonstrated by screening mutant strains of Streptococcus mutans to identify the enzyme(s) responsible for synthesizing the tRNA position 37 modification threonylcarbamoyladenosine (t6 A).

Summary Argonaute (AGO) proteins and small RNAs (sRNAs) are core components of the RNA‐induced silencing complex (RISC). It has been reported that miRNAs regulate plant height and grain size in rice, but which AGO is involved in grain size regulation remains unclear. Here, we report that enhanced expression of OsAGO17, a putative AGO protein, could improve grain size and weight and promote stem development in rice. Cytological evidence showed that these effects are mainly caused by alteration of cell elongation. Expression analyses showed that OsAGO17 was highly expressed in young panicles and nodes, which was consistent with the expression pattern of OsmiR397b. SRNA sequencing, stem‐loop RT‐PCR and sRNA blotting showed that the expression of OsmiR397b was reduced in ago17 and enhanced in the OsAGO17 OE lines. Four OsmiR397b target laccase (LAC) genes showed complementary expression patterns with OsAGO17 and OsmiR397b. Combined with the results of immunoprecipitation (IP) analysis, we suggested that OsAGO17 formed an RISC with OsmiR397b and affected rice development by suppression of LAC expression. In conclusion, OsAGO17 might be a critical protein in the sRNA pathway and positively regulates grain size and weight in rice.

Inverse electron‐demand Diels–Alder cycloadditions (iEDDAC) between tetrazines and strained alkenes/alkynes have emerged as essential tools for studying and manipulating biomolecules. A light‐triggered version of iEDDAC (photo‐iEDDAC) is presented that confers spatio‐temporal control to bioorthogonal labeling in vitro and in cellulo. A cyclopropenone‐caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo‐DMBO) was designed that is unreactive towards tetrazines before light‐activation, but engages in iEDDAC after irradiation at 365 nm. Aminoacyl tRNA synthetase/tRNA pairs were discovered for efficient site‐specific incorporation of tetrazine‐containing amino acids into proteins in living cells. In situ light activation of photo‐DMBO conjugates allows labeling of tetrazine‐modified proteins in living E. coli. This allows proteins in living cells to be modified in a spatio‐temporally controlled manner and may be extended to photo‐induced and site‐specific protein labeling in animals., Light‐induced protein labeling: Cyclopropenone‐caged dibenzoannulated bicyclononynes (photo‐DMBO) are photo‐activatable dienophiles that engage in rapid inverse electron‐demand Diels–Alder cycloadditions with tetrazines upon light‐induced decarbonylation. Site‐specific incorporation of methyl‐tetrazine amino acids allows photo‐induced protein labeling in living cells with spatio‐temporal control using photo‐DMBO fluorophore conjugates.

During ribosome translocation, the elongation factor EF‐G undergoes large conformational change while maintaining its contact with the moving tRNA. We previously measured a power stroke accompanying EF‐G catalysis, which was consistent with structural studies. However, the role of power stroke in translocation fidelity remains unclear. Here, we report quantitative measurements of the power strokes of structurally modified EF‐Gs by using two different techniques and reveal the correlation between power stroke and translocation efficiency and fidelity. We discovered that the reduced power stroke only lowered the percentage of translocation but did not introduce translocation error. The established force ‐structure–function correlation for EF‐G indicates that power stroke drives ribosomal translocation, but the mRNA reading frame is probably maintained by ribosome itself. Furthermore, the microscope detection method reported here can be simply implemented for other biochemical applications., Power stroke: The ribosome translocase EF‐G generates substantial power stroke during translocation when its conformational change is not restricted. A short crosslinker restricting the conformational change will reduce this force by nearly 30 pN. The reduced power stroke decreases the translocation efficiency but does not introduce translocation error.

The characterization of a complete mitogenome is widely used in genomics studies for systematics and evolutionary research. However, the sequences and structural motifs contained within the mitogenome of Testudines taxa have rarely been examined. The present study decodes the first complete mitochondrial genome of the Indian Tent Turtle, Pangshura tentoria (16,657 bp) by using next‐generation sequencing. This denovo assembly encodes 37 genes: 13 protein‐coding genes (PCGs), 22 transfer RNA (tRNAs), two ribosomal RNA, and one control region (CR). Most of the genes were encoded on majority strand, except for one PCG (NADH dehydrogenase subunit 6) and eight tRNAs. Most of the PCGs were started with an ATG initiation codon, except for Cytochrome oxidase subunit 1 with “GTG” and NADH dehydrogenase subunit 5 with “ATA.” The termination codons, “TAA” and “AGA” were observed in two subunits of NADH dehydrogenase gene. The relative synonymous codon usage analysis revealed the maximum abundance of alanine, isoleucine, leucine, and threonine. The nonsynonymous/synonymous ratios were, The present study decodes the first complete mitochondrial genome of the Indian Tent Turtle, Pangshura tentoria by using next‐generation sequencing. This denovo assembly encodes 37 genes: 13 protein coding genes (PCGs), 22 transfer RNA (tRNAs), two ribosomal RNA (rRNAs), and one control region (CR). The maximum‐likelihood (ML) phylogenetic trees using concatenation of 13 PCGs revealed the close relationships of P. tentoria with Batagur trivittata.

The essential type of endonuclease that removes 5' leader sequences from transfer RNA precursors is called RNase P. While ribonucleoprotein RNase P enzymes containing a ribozyme are found in all domains of life, another type of RNase P called 'PRORP', for 'PROtein-only RNase P', is composed of protein that occurs only in a wide variety of eukaryotes, in organelles and in the nucleus. Here, to find how PRORP functions integrate with other cell processes, we explored the protein interaction network of PRORP1 in Arabidopsis mitochondria and chloroplasts. Although PRORP proteins function as single subunit enzymes in vitro, we found that PRORP1 occurs in protein complexes and is present in high-molecular-weight fractions that contain mitochondrial ribosomes. The analysis of immunoprecipitated protein complexes identified proteins involved in organellar gene expression processes. In particular, direct interaction was established between PRORP1 and MNU2 a mitochondrial nuclease. A specific domain of MNU2 and a conserved signature of PRORP1 were found to be directly accountable for this protein interaction. Altogether, results revealed the existence of an RNA maturation complex in Arabidopsis mitochondria and suggested that PRORP proteins cooperated with other gene expression factors for RNA maturation in vivo.

RNA can be modified in over 100 distinct ways, and these modifications are critical for function. Pseudouridine synthases catalyse pseudouridylation, one of the most prevalent RNA modifications. Pseudouridine synthase 7 modifies a variety of substrates in Saccharomyces cerevisiae including tRNA, rRNA, snRNA, and mRNA, but the substrates for other budding yeast Pus7 homologues are not known. We used CRISPR‐mediated genome editing to disrupt Candida albicans PUS7 and find absence leads to defects in rRNA processing and a decrease in cell surface hydrophobicity. Furthermore, C. albicans Pus7 absence causes temperature sensitivity, defects in filamentation, altered sensitivity to antifungal drugs, and decreased virulence in a wax moth model. In addition, we find C. albicans Pus7 modifies tRNA residues, but does not modify a number of other S. cerevisiae Pus7 substrates. Our data suggests C. albicans Pus7 is important for fungal vigour and may play distinct biological roles than those ascribed to S. cerevisiae Pus7.

Nucleotides of transfer RNAs (tRNAs) are highly modified, particularly at the anticodon. Bacterial tRNAs that read A-ending codons are especially notable. The U34 nucleotide canonically present in these tRNAs is modified by a wide range of complex chemical constituents. An additional two A-ending codons are not read by U34-containing tRNAs but are accommodated by either inosine or lysidine at the wobble position (I34 or L34). The structural basis for many N34 modifications in both tRNA aminoacylation and ribosome decoding has been elucidated, and evolutionary conservation of modifying enzymes is also becoming clearer. Here we present a brief review of the structure, function, and conservation of wobble modifications in tRNAs that translate A-ending codons. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1158-1166, 2019.

Phage display is a powerful approach for evolving proteins and peptides with new functions, but the properties of the molecules that can be evolved are limited by the chemical diversity encoded. Herein, we report a system for incorporating non‐canonical amino acids (ncAAs) into proteins displayed on phage using the pyrrolysyl‐tRNA synthetase/tRNA pair. We improve the efficiency of ncAA incorporation using an evolved orthogonal ribosome (riboQ1), and encode a cyclopropene‐containing ncAA (CypK) at diverse sites on a displayed single‐chain antibody variable fragment (ScFv), in response to amber and quadruplet codons. CypK and an alkyne‐containing ncAA are incorporated at distinct sites, enabling the double labeling of ScFv with distinct probes, through mutually orthogonal reactions, in a one‐pot procedure. These advances expand the number of functionalities that can be encoded on phage‐displayed proteins and provide a foundation to further expand the scope of phage display applications.

Transfer RNAs are among the most ancient molecules of life on earth. Beyond their crucial role in protein synthesis as carriers of amino acids, they are also important players in a plethora of other biological processes. Many debates in term of biogenesis, regulation and function persist around these fascinating non-coding RNAs. Our review focuses on the first step of their biogenesis in eukaryotes, i.e. their transcription from nuclear genes. Numerous and complementary ways have emerged during evolution to regulate transfer RNA gene transcription. Here, we will summarize the different actors implicated in this process: cis-elements, trans-factors, genomic contexts, epigenetic environments and finally three-dimensional organization of nuclear genomes. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1099-1108, 2019.

Some proteins are expressed as a result of a ribosome frameshifting event that is facilitated by a slippery site and downstream secondary structure elements in the mRNA. This review summarizes recent progress in understanding mechanisms of –1 frameshifting in several viral genes, including IBV 1a/1b, HIV‐1 gag‐pol, and SFV 6K, and in Escherichia coli dnaX. The exact frameshifting route depends on the availability of aminoacyl‐tRNAs: the ribosome normally slips into the –1‐frame during tRNA translocation, but can also frameshift during decoding at condition when aminoacyl‐tRNA is in limited supply. Different frameshifting routes and additional slippery sites allow viruses to maintain a constant production of their key proteins. The emerging idea that tRNA pools are important for frameshifting provides new direction for developing antiviral therapies.

T-box riboswitches are a widespread class of structured noncoding RNAs in Gram-positive bacteria that regulate the expression of amino acid-related genes. They form negative feedback loops to maintain steady supplies of aminoacyl-transfer RNAs (tRNAs) to the translating ribosomes. T-box riboswitches are located in the 5' leader regions of mRNAs that they regulate and directly bind to their cognate tRNA ligands. T-boxes further sense the aminoacylation state of the bound tRNAs and, based on this readout, regulate gene expression at the level of transcription or translation. T-box riboswitches consist of two conserved domains-a 5' Stem I domain that is involved in specific tRNA recognition and a 3' antiterminator/antisequestrator (or discriminator) domain that senses the amino acid on the 3' end of the bound tRNA. Interaction of the 3' end of an uncharged but not charged tRNA with a thermodynamically weak discriminator domain stabilizes it to promote transcription readthrough or translation initiation. Recent biochemical, biophysical, and structural studies have provided high-resolution insights into the mechanism of tRNA recognition by Stem I, several structural models of full-length T-box-tRNA complexes, mechanism of amino acid sensing by the antiterminator domain, as well as kinetic details of tRNA binding to the T-box riboswitches. In addition, translation-regulating T-box riboswitches have been recently characterized, which presented key differences from the canonical transcriptional T-boxes. Here, we review the recent developments in understanding the T-box riboswitch mechanism that have employed various complementary approaches. Further, the regulation of multiple essential genes by T-boxes makes them very attractive drug targets to combat drug resistance. The recent progress in understanding the biochemical, structural, and dynamic aspects of the T-box riboswitch mechanism will enable more precise and effective targeting with small molecules. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1167-1180, 2019.

Transcription is the fundamental process that enables the expression of genetic information. DNA-directed RNA polymerase (RNAP) uses one strand of the DNA duplex as template to produce complementary RNA molecules that serve in translation (rRNA, tRNA), protein synthesis (mRNA), and regulation (sRNA). Although the RNAP core is catalytically competent for RNA synthesis, the selectivity of transcription initiation requires a sigma (σ) factor for promoter recognition and opening. Expression of alternative σ factors provides a powerful mechanism to control the expression of discrete sets of genes (a σ regulon) in response to specific nutritional, developmental, or stress-related signals. Here, I review the key insights that led to the original discovery of σ factor 50 years ago and the subsequent discovery of alternative σ factors as a ubiquitous mechanism of bacterial gene regulation. These studies form a prelude to the more recent, genomics-enabled insights into the vast diversity of σ factors in Bacteria.

Translation is the most error-prone process in protein synthesis; however, it is important that accuracy is maintained because erroneous translation has been shown to affect all domains of life. Translational quality control is maintained by both proteins and RNA through intricate processes. The aminoacyl-tRNA synthetases help maintain high levels of translational accuracy through the esterification of tRNA and proofreading mechanisms. tRNA is often recognized by an aminoacyl-tRNA synthetase in a sequence and structurally dependent manner, sometimes involving modified nucleotides. Additionally, some proofreading mechanisms of aminoacyl-tRNA synthetases require tRNA elements for hydrolysis of a noncognate aminoacyl-tRNA. Finally, tRNA is also important for proper decoding of the mRNA message by codon and anticodon pairing. Here, recent developments regarding the importance of tRNA in maintenance of translational accuracy are reviewed. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1150-1157, 2019.

tRNA(His) guanylyltransferase (Thg1) specifies eukaryotic tRNA(His) identity by catalyzing a 3’ to 5’ non-Watson Crick (WC) addition of guanosine to the 5’-end of tRNA(His). Thg1 family enzymes in Archaea and Bacteria, called Thg1-like-proteins (TLPs), catalyze a similar but distinct 3’ to 5’ addition in an exclusively WC-dependent manner. Here, a genetic system in Saccharomyces cerevisiae was employed to further assess biochemical differences between Thg1 and TLPs. Utilizing a novel 5’-end sequencing pipeline, we find that a Bacillus thuringiensis TLP sustains growth of a thg1Δ strain by maintaining a WC-dependent addition of U(−1) across from A(73). Additionally, we observe 5’-end heterogeneity in S. cerevisiae small nucleolar RNAs (snoRNAs), an observation that may inform methods of annotation and mechanisms of snoRNA processing.

The emergence of multidrug-resistant bacteria necessitates the identification of unique targets of intervention and compounds that inhibit their function. Gram-positive bacteria use a well-conserved tRNA-responsive transcriptional regulatory element in mRNAs, known as the T-box, to regulate the transcription of multiple operons that control amino acid metabolism. T-box regulatory elements are found only in the 5'-untranslated region (UTR) of mRNAs of Gram-positive bacteria, not Gram-negative bacteria or the human host. Using the structure of the 5'UTR sequence of the Bacillus subtilis tyrosyl-tRNA synthetase mRNA T-box as a model, in silico docking of 305 000 small compounds initially yielded 700 as potential binders that could inhibit the binding of the tRNA ligand. A single family of compounds inhibited the growth of Gram-positive bacteria, but not Gram-negative bacteria, including drug-resistant clinical isolates at minimum inhibitory concentrations (MIC 16-64 μg mL-1 ). Resistance developed at an extremely low mutational frequency (1.21×10-10 ). At 4 μg mL-1 , the parent compound PKZ18 significantly inhibited in vivo transcription of glycyl-tRNA synthetase mRNA. PKZ18 also inhibited in vivo translation of the S. aureus threonyl-tRNA synthetase protein. PKZ18 bound to the Specifier Loop in vitro (Kd ≈24 μm). Its core chemistry necessary for antibacterial activity has been identified. These findings support the T-box regulatory mechanism as a new target for antibiotic discovery that may impede the emergence of resistance.