Amino acid availability in Gram-positive bacteria is monitored by T-box riboswitches. T-boxes directly bind tRNAs, assess their aminoacylation state, and regulate the transcription or translation of downstream genes to maintain nutritional homeostasis. Here, we report co-crystal and cryo-EM structures of Geobacillus kaustophilus and Bacillus subtilis T-box-tRNA complexes detailing their Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Ribosomal RNA (rRNA) biogenesis takes place in the nucleolus, the most prominent condensate of the eukaryotic nucleus. The proper assembly and integrity of the nucleolus reflects the accurate synthesis and processing of rRNAs which in turn, as major components of ribosomes, ensure the uninterrupted flow of the genetic information during translation. Therefore, the abundant production of rRNAs in a precisely functional nucleolus is of outmost importance for the cell viability and requires the concerted action of essential enzymes, associated factors and epigenetic marks. The coordination and regulation of such an elaborate process depends on not only protein factors, but also on numerous regulatory non-coding RNAs (ncRNAs). Herein, we focus on RNA-mediated mechanisms that control the synthesis, processing and modification of rRNAs in mammals. We highlight the significance of regulatory ncRNAs in rRNA biogenesis and the maintenance of the nucleolar morphology, as well as their role in human diseases and as novel druggable molecular targets.

Riboswitches are structured non-coding RNAs found in the 5′ UTR of important genes for bacterial metabolism, virulence and survival. Upon the binding of specific ligands that can vary from simple ions to complex molecules such as nucleotides and tRNAs, riboswitches change their local and global mRNA conformations to affect downstream transcription or translation. Due to their dynamic nature and central regulatory role in bacterial metabolism, riboswitches have been exploited as novel RNA-based targets for the development of new generation antibacterials that can overcome drug-resistance problems. During recent years, several important riboswitch structures from many bacterial representatives, including several prominent human pathogens, have shown that riboswitches are ideal RNA targets for new compounds that can interfere with their structure and function, exhibiting much reduced resistance over time. Most interestingly, mainstream antibiotics that target the ribosome have been shown to effectively modulate the regulatory behavior and capacity of several riboswitches, both in vivo and in vitro, emphasizing the need for more in-depth studies and biological evaluation of new antibiotics. Herein, we summarize the currently known compounds that target several main riboswitches and discuss the role of mainstream antibiotics as modulators of T-box riboswitches, in the dawn of an era of novel inhibitors that target important bacterial regulatory RNAs.

T-box riboswitches (T-boxes) are essential RNA regulatory elements with a remarkable structural diversity, especially among bacterial pathogens. In staphylococci, all glyS T-boxes synchronize glycine supply during synthesis of nascent polypeptides and cell wall formation and are characterized by a conserved and unique insertion in their antiterminator/terminator domain, termed stem Sa. Interestingly, in Staphylococcus aureus the stem Sa can accommodate binding of specific antibiotics, which in turn induce robust and diverse effects on T-box-mediated transcription. In the present study, domain swap mutagenesis and probing analysis were performed to decipher the role of stem Sa. Deletion of stem Sa significantly reduces both the S. aureus glyS T-box-mediated transcription readthrough levels and the ability to discriminate among tRNAGly isoacceptors, both in vitro and in vivo. Moreover, the deletion inverted the previously reported stimulatory effects of specific antibiotics. Interestingly, stem Sa insertion in the terminator/antiterminator domain of Geobacillus kaustophilus glyS T-box, which lacks this domain, resulted in elevated transcription in the presence of tigecycline and facilitated discrimination among proteinogenic and nonproteinogenic tRNAGly isoacceptors. Overall, stem Sa represents a lineage-specific structural feature required for efficient staphylococcal glyS T-box-mediated transcription and it could serve as a species-selective druggable target through its ability to modulate antibiotic binding.

No abstract