Functional and structural analysis of tRNA-mediated gene regulation in pathogens
Loading...
Date
2023-07-25
Authors
Γιαρίμογλου, Νικολέτα
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
tRNA-mediated regulation of transcription is widespread among Gram-positive bacteria and involves specific RNA-RNAs interactions which sense both charged and uncharged tRNAs. This intricate mechanism relies on the interaction between tRNA molecules, and the T-box regulatory leader element, termed T-box riboswitch, at the 5' untranslated region (UTR) of mRNA molecules. This interaction controls the expression of a wide array of genes responsible for amino acid transport, biosynthesis, and tRNA aminoacylation. Through a conformational switch triggered by tRNA binding, T-box riboswitches flip between two distinct conformations known as “terminator/ sequestrator” and “antiterminator/ antisequestrator” and allow or stall transcription or translation of the downstream genes under control. The T-box:tRNA mediated regulatory mechanism involves the initial recognition of the cognate tRNA molecule by the Specifier loop within the Stem I region, through a codon-anticodon like interaction. This contact is further stabilised by additional contacts of the tRNA elbow and the apical loop, which in turn depends on the AG bulge of Stem I. Finally, the T-box interacts with the tRNA 3′ end with a “discriminator” structure and interrogates its aminoacylation status using a steric sieve provided by a conserved wobble base-pair. In the absence of aminoacylation the transcription or translation is allowed. The presence of an amino acid on the other hand, interferes with tRNA-T-box stacking and inhibits gene expression. T-boxes are dynamic regulatory elements that control several essential genes for bacterial metabolism, and therefore represent promising RNA targets for the development of novel antibacterials to combat drug resistance.
In staphylococci, the glyS T-box interacts with five tRNAGly isoacceptors to regulate glycine supply during synthesis of nascent proteins, as well as the synthesis of pentaglycine peptides that stabilize cell wall formation. This T-box is characterized by a distinct and conserved insertion within its antiterminator/terminator domain, known as Stem Sa. Notably in Staphylococcus aureus, Stem Sa region can interact with specific antibiotics and affects T-box-mediated transcription. The significance of Stem Sa was investigated by domain swap mutagenesis and probing analyses. The deletion of Stem Sa resulted in a significant reduction in the S. aureus glyS T-box- mediated transcription and abolished the ability to discriminate among different tRNAGly isoacceptors, both in vitro and in vivo. Additionally, the deletion inverted the previously observed stimulatory effects of tigecycline on transcription. However, the introduction of Stem Sa in Geobacillus kaustophilus glyQ T-box, which naturally lacks this domain, led to enhanced transcription in the presence of tigecycline and the presence of Stem Sa facilitated discrimination between proteinogenic and nonproteinogenic tRNAGly isoacceptors. Overall, it was shown Stem Sa represents a lineage-specific structural feature which distinguishes glyS T-boxes in staphylococci from other species and is essential for the proper function of S. aureus glyS T-box.
To get insights on the biological role of the tRNAGly isoacceptor genes redundancy in S. aureus, the CRISPR/Cas9 genome editing tool was used to knock out P1 tRNAGlyGCC and NP tRNA genes cluster. Notably, depletion of the NP tRNA cluster was found to be lethal. In contrast, the depletion of P1 tRNAGly did not have any noticeable impact on the growth rate of S. aureus, although the edited strain exhibited increased susceptibility to antibiotics targeting the cell wall. Transcriptomic analysis of the edited strain revealed downregulation of genes implicated in the biosynthesis of amino acids and vitamins, as well as differentially expression of genes required for the iron homeostasis. Also, several crucial genes involved in cell wall formation and antibiotic resistance were significantly downregulated. These suggest that tRNAs are important regulators of bacterial homeostasis and reveals the tRNA-mediated interconnection of distinct cellular processes.
Moreover, to investigate their role in the correct folding and proper function of T-box riboswitches, point mutations were introduced at conserved nucleotides that participate in the “discriminator” structure of G. kaustophilus glyQ T-box. In addition, structural and functional analysis of the glycyl T-boxes from four representative human pathogens Clostridium tetani, Listeria monocytogenes, Streptococcus pneumoniae and Streptococcus pyogenes was performed and showed that despite their structural peculiarities, all four T-boxes responded to glycine starvation in vivo. Subsequent probing analysis revealed that the secondary structures of streptococcal T-boxes deviate from the other two which follow the typical T-box structural pattern. The K-turn motif observed absent from both streptococcal T-boxes, the S. pneumoniae glyQS T-box bears an unusually short linker and notably the S. pyogenes glyQS T-box contains a Stem II domain that is typically absent from glycyl T-boxes.
Finally, a synthetic compound that specifically targets the apical loop of stem I was identified after in silico analysis and in vitro and in vivo screening. The compound, termed arrêT-box, inhibits S. aureus growth by specifically targeting T-box riboswitches and more importantly, does not induce resistance. Transcriptomic analysis of cultures in the presence of the compound revealed that affects several crucial cellular processes such as translation, several biosynthetic processes and stress response, and represents a promising leading candidate for the development of novel antibacterial compounds.
In conclusion, the present thesis provides novel information regarding the structure and function of T-box riboswitches from different bacteria with an emphasis in pathogens. The results support and highlight the diversity of structural features among different T-boxes that mirror the variation in metabolic adaptation among pathogens. Finally, the broader characterization of several T-boxes from a variety of representative pathogenic bacteria is essential and guides the development and optimization of novel antibiotics that target T-boxes.
Description
Keywords
T-box riboswitches, tRNA, Transcription regulation, Bacterial pathogens, Antibiotics