A glyS T-box riboswitch with species-specific structural features responding to both proteinogenic and nonproteinogenic tRNA^Gly isoacceptors

Abstract

In Staphylococcus aureus, a T-box riboswitch exists upstream of the glyS gene to regulate transcription of the sole glycyl-tRNA synthetase, which aminoacylates five tRNA Gly isoacceptors bearing GCC or UCC anticodons. Subsequently, the glycylated tRNAs serve as substrates for decoding glycine codons during translation, and also as glycine donors for exoribosomal synthesis of pentaglycine peptides during cell wall formation. Probing of the predicted T-box structure revealed a long stem I, lacking features previously described for similar T-boxes. Moreover, the antiterminator stem includes a 42-nt long intervening sequence, which is staphylococci-specific. Finally, the terminator conformation adopts a rigid two-stem structure, where the intervening sequence forms the first stem followed by the second stem, which includes the more conserved residues. Interestingly, all five tRNA Gly isoacceptors interact with S. aureus glyS T-box with different binding affinities and they all induce transcription readthrough at different levels. The ability of both GCC and UCC anticodons to interact with the specifier loop indicates ambiguity during the specifier triplet reading, similar to the unconventional reading of glycine codons during protein synthesis. The S. aureus glyS T-box structure is consistent with the recent crystallographic and NMR studies, despite apparent differences, and highlights the phylogenetic variability of T-boxes when studied in a genome-dependent context. Our data suggest that the S. aureus glyS T-box exhibits differential tRNA selectivity, which possibly contributes toward the regulation and synchronization of ribosomal and exoribosomal peptide synthesis, two essential but metabolically unrelated pathways.

Unboxing the T‐box riboswitches—A glimpse into multivalent and multimodal RNA–RNA interactions

“…Notably, while most translational T-boxes lack the long Stem I's, they frequently sport additional insertions in their discriminator domains, such as in Stem III and especially in Helix A2 (i.e., on top of the antiterminator). These features may aid the folding of the discriminator helical elements and facilitate their refolding between the conformers, or even make additional contacts to the tRNA acceptor stem, as proposed for the Stem Sa in the Staphylococci glyQ Tboxes (Apostolidi et al, 2015;Stamatopoulou et al, 2017).…”

Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus

“…To address this hypothesis, we cloned the unlinked genes encoding thioredoxin (trxA) or thioredoxin reductase (trxB) on multicopy plasmids and placed the genes under the control of the T box riboswitch of the glycyl-tRNA synthetase gene promoter PglyS (SAOUHSC_01666) (44,46). This promoter is not known to be regulated by Spx and would be expected to exert housekeeping levels of gene expression, since S. aureus encodes only one glycyl-tRNA synthetase.…”

Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors

“…Recent studies focused on the characterization of T-boxes from pathogenic bacteria revealed unexpected complexities of T-box systems both at the structural and biochemical level. T-boxes are no longer considered of single tRNA specificity and can contain species-specific structural features, like SL with tandem or overlapping codons ( 22 , 23 ). Certain T-boxes, exemplified by those in Actinobacteria, control translation initiation instead of transcription, in comparable genomic contexts ( 24 ).…”