A dual T-box riboswitch in Enterococcus faecium binds two tRNAs
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ABSTRACT:
PROVIDER: PRJNA678524 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13062392.fastq.gz | Fastqsanger.gz | |||
| SRR13062393.fastq.gz | Fastqsanger.gz | |||
| SRR13062394.fastq.gz | Fastqsanger.gz | |||
| SRR13062395.fastq.gz | Fastqsanger.gz | |||
| SRR13062396.fastq.gz | Fastqsanger.gz |
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Project description:A set of small RNAs was identified in Vancomycin-resistant Enterococcus faecium, a leading cause of MDR infections. We described here the function of srn_2050, acting as a T-box riboswitch to regulate expression of downstream genes encoding the HisRS and AspRS aminoacyl-tRNA synthetases. Comparative RNAseq between Aus0004 and isogenic srn_2050 mutant identified the genes whose expression is impacted by the RNA. srn_2050 structure in its ‘off state’ was deciphered by in-line probing, containing T-box consensus sequences, a pseudoknot, a specifier loop and a terminator. Transcription binding assays between the riboswitch and either tRNAAsp or tRNAHis indicate that each deacylated tRNA interacts with the T-box. Their anticodons bind to a GACAC sequence within the specifier loop (GAC and CAC are Asp and His codons, respectively), whereas tRNATyr (UA/C-U) does not. A pioneering evaluation of E. faecium amino acid auxotrophy, with emphasis on E. faecium strain Aus0004, revealed auxotrophy for Histidine but not for Aspartic acid. Based on comparative growths and RNAseq between Aus004 and Aus004-srn2050, the riboswitch is shown essential for growth under aspartate starvation. This is the first example of a functional riboswitch in E. faecium with two overlapping codons allowing a dual tRNA-dependent regulation at transcriptional level.
2020-11-16 | GSE161499 | GEO
Project description:In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5' untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here, we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is a target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5'-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3'-end. Corresponding variations in protein levels led us to hypothesize that coordinated RNA decay represents another level in the hierarchical methionine biosynthesis control network to adjust methionine biosynthesis enzyme amounts to current metabolic requirements.
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Project description:In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5' untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here, we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is a target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5'-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3'-end. Corresponding variations in protein levels led us to hypothesize that coordinated RNA decay represents another level in the hierarchical methionine biosynthesis control network to adjust methionine biosynthesis enzyme amounts to current metabolic requirements.
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Project description: Not available
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Project description:T-box riboswitches belong to a specific class of RNA regulatory elements that control gene expression in Gram-positive bacteria, including prominent human pathogens. They sense the availability of amino acids by detecting the aminoacylation status of their cognate tRNAs and regulate the expression of genes involved in aminoacylation, amino acid transport, and metabolism. Recent advances on the structures and mechanisms of several regulatory non-coding RNAs among pathogenic bacteria have garnered attention for the development of a new generation of species-specific antibacterials. The frequently acquired resistance against current antibiotics has emerged as a significant challenge for healthcare systems and a serious threat to public health. Herein, we report the characterization of an effective T-box riboswitch inhibitor, termed T-box-i, which efficiently disrupts T-box riboswitch-mediated transcription in vivo. T-box-i was selected through a virtual screening campaign of commercially available small molecules against high-resolution crystallographic structures of T-box riboswitches. It exhibited no cytotoxicity in mammalian cells nor induced antibiotic resistance in S. aureus cultures. These findings provide valuable insights into exploiting T-box riboswitches as antibiotic targets and underscore the therapeutic potential of compounds that selectively target extensively structured regulatory RNA elements and interfaces to combat drug-resistant pathogens.
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Project description:We show that DANCE-MaP permits measurement of state-specific per-nucleotide reactivities, direct secondary structure PAIRs, and tertiary RINGs for RNA structural ensembles. Here, we demonstrate DANCE-MaP on the V. vulnificus add riboswitch.
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Project description:We used MIST (Microarray Identification of Shifted tRNAs), a previously established in vitro approach, to systematically assess the specificity of complexes between native H. sapiens tRNAs and recombinant P. falciparum tRip. We demonstrate that tRip unexpectedly binds to host tRNAs with a wide range of specificities, suggesting that only a small subset of human tRNAs are preferentially imported into the parasite.
2021-07-07 | GSE179567 | GEO
Project description:Redundancy in the number of tRNA genes which occur across species is poorly understood and, in many cases, remains essentially unexplored. In Staphylococcus aureus, several tRNAGly genes encode isoacceptors that either participate in protein synthesis or cell wall formation, upon aminoacylation by the sole glycyl-tRNA synthetase (GlyRS). Transcription of GlyRS, is regulated by a glyS T-box riboswitch in the 5’UTR, which responds to all tRNAGly isoacceptors and exhibits species-specific RNA conformations. To address whether disruption of the tRNAGly expression impacts the balance between ribosomal translation and cell wall formation, we used CRISPR/Cas9 editing to ablate one proteinogenic tRNAGly gene copy which is the stronger ligand of the glyS T-box riboswitch. Interestingly, no discernible impact on the growth and translational activity of S. aureus was observed, suggesting that the remaining tRNAs could make up for this deficiency. However, transcriptomics and proteomics analyses revealed that the edited strain had deficient cell wall integrity, and subsequent experimentation showed increased susceptibility to antibiotics targeting the cell wall. Interestingly, the observed alteration in the proteome level were independent of the glycine codon usage. Moreover, the edited strain exhibited reduced biofilm formation but retained its ability to invade human cell cultures. Overall, the present study highlights the important role of tRNA gene copy redundancy in the physiology of an important pathogen like S. aureus and consolidates the regulatory role of tRNAs in metabolic homeostasis.
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Project description:Plant basic helix-loop-helix (bHLH) transcription factors are involved in physiological and developmental processes, and also play essential roles in abiotic stresses. However, their exact roles in abiotic stress are still need to be elucidated, and most of bHLHs have not been functionally characterized. In the present study, we characterized the functional role of AtbHLH112 in response to abiotic stresses. AtbHLH112 is a nuclear-localized protein, and its nuclear-localization is induced by salt, drought and ABA. Besides binding to E-box motif, AtbHLH112 is found to bind to a novel motif with the sequence M-bM-^@M-^\GG[GT]CC[GT][GA][TA]CM-bM-^@M-^] (GCG-box), and the binding affinity is induced by salt and ABA. Gain- and loss-of-function analyses showed that the transcript level of AtbHLH112 is positively correlated with salt and drought tolerance. AtbHLH112 mediates stress tolerance by upregulating the expression of P5CS genes and decreasing the expression of P5CDH and PRODH genes to increase proline levels, and via enhancing the expression of POD and SOD genes to improve ROS scavenging ability. All data together suggested that AtbHLH112 regulates the expression of genes through binding to GCG-box and E-box to mediate the physiological stress responses, including proline biosynthesis and ROS scavenging pathways to enhance stress tolerance. Differentially expression genes of AtbHLH112-overexpression plants, mutant (SALK_033618C) plants and wild type of Columbia Arabidopsis thaliana were measured under salt stressed and normal condition for 3 hours, respectively. Three independent experiments were performed at each treatment using different plants for each experiment.
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