Essentiality of threonylcarbamoyladenosine (t⁶A), a universal tRNA modification, in bacteria

Abstract

Threonylcarbamoyladenosine (t6A) is a modified nucleoside universally conserved in tRNAs in all three kingdoms of life. The recently discovered genes for t6A synthesis, including tsaC and tsaD, are essential in model prokaryotes but not essential in yeast. These genes had been identified as antibacterial targets even before their functions were known. However, the molecular basis for this prokaryotic-specific essentiality has remained a mystery. Here, we show that t6A is a strong positive determinant for aminoacylation of tRNA by bacterial-type but not by eukaryotic-type isoleucyl-tRNA synthetases and might also be a determinant for the essential enzyme tRNAIle-lysidine synthetase. We confirm that t6A is essential in Escherichia coli and a survey of genome-wide essentiality studies shows that genes for t6A synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t6A is dispensable in Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803 and Streptococcus mutans. Proteomic analysis of t6A- D. radiodurans strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t6A in tRNAs. Thus, although t6A is universally conserved in tRNAs, its role in translation might vary greatly between organisms.

Transcriptome-wide Analysis of Roles for tRNA Modifications in Translational Regulation

“…However, for two mutants – bud32 Δ and gon7 Δ, both members of the conserved KEOPS complex (Daugeron et al, 2011; Downey et al, 2006; Kisseleva-Romanova et al, 2006; Srinivasan et al, 2011) – we repeatedly obtained haploid strains bearing an additional copy of ChrIX, suggesting that the KEOPS complex may be essential in our strain background. We included these mutants in the final set of 57 mutants despite this aneuploidy, as many of the dramatic ribosome footprinting phenotypes observed in these two mutants were also observed to a lesser extent in the euploid cgi121 Δ mutant (which exhibits partial but incomplete loss of the t 6 A modification (Thiaville et al, 2015)), suggesting that many of the observed phenotypes accurately reflect KEOPS function. Nonetheless, we urge caution in interpreting results obtained from bud32 Δ and gon7 Δ cells, as observed phenotypes may be secondary to second-site mutations.…”

Factors That Shape Eukaryotic tRNAomes: Processing, Modification and Anticodon–Codon Use

“…In some species, t 6 A 37 exists as is whereas in others it is in a cyclic form, ct 6 A 37 (see [101,123]) or a hypermodified form thereof, hereafter referred to collectively for simplicity as t 6 A 37 [124,125]. As t 6 A 37 is found almost without exception on all tRNAs that decode the sixteen ANN codons, it is a most pervasive anticodon loop modification (see [101,123,126]).…”

“…Furthermore, i 6 A 37 is absent on tRNAs for Cys, Tyr and Trp in a species-specific manner such that budding yeast, fission yeast and human cells each contain distinct subsets of i 6 A37-containing tRNAs [127]; in cases where these tRNAs lack i 6 A 37 they usually have an encoded G at 37, found as m 1 G in the tRNA [128,129] (Figure 5A). Again, this variability is in stark contrast to the omnipresence of t 6 A 37 on tRNAs that read ANN codons (see [101,123,126]).…”

Integrative analyses of the RNA modification machinery reveal tissue- and cancer-specific signatures

“…The second most frequently up-regulated RMP was the L antigen family member 3 (LAGE3), a component of complex responsible for formation of N6-threonylcarbamoyladenosine (t 6 A) in position 37 of tRNAs ( Figure 4D). Interestingly, this modification is found in the anticodon stem-loop of many tRNAs decoding ANN codons [72], and has been shown to affect both translation accuracy as well as efficiency [73].…”