<jats:title>Summary</jats:title><jats:p>Threonylcarbamoyladenosine (t<jats:sup>6</jats:sup><jats:styled-content style="fixed-case">A</jats:styled-content>) is a modified nucleoside universally conserved in t<jats:styled-content style="fixed-case">RNA</jats:styled-content>s in all three kingdoms of life. The recently discovered genes for t<jats:sup>6</jats:sup><jats:styled-content style="fixed-case">A</jats:styled-content> synthesis, including <jats:italic>tsa</jats:italic><jats:styled-content style="fixed-case"><jats:italic>C</jats:italic></jats:styled-content> and <jats:italic>tsa</jats:italic><jats:styled-content style="fixed-case"><jats:italic>D</jats:italic></jats:styled-content>, 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 t<jats:sup>6</jats:sup><jats:styled-content style="fixed-case">A</jats:styled-content> is a strong positive determinant for aminoacylation of t<jats:styled-content style="fixed-case">RNA</jats:styled-content> by bacterial‐type but not by eukaryotic‐type isoleucyl‐t<jats:styled-content style="fixed-case">RNA</jats:styled-content> synthetases and might also be a determinant for the essential enzyme t<jats:styled-content style="fixed-case">RNA</jats:styled-content><jats:sup>Ile</jats:sup>‐lysidine synthetase. We confirm that t<jats:sup>6</jats:sup><jats:styled-content style="fixed-case">A</jats:styled-content> is essential in <jats:styled-content style="fixed-case"><jats:italic>E</jats:italic></jats:styled-content><jats:italic>scherichia coli</jats:italic> and a survey of genome‐wide essentiality studies shows that genes for t<jats:sup>6</jats:sup><jats:styled-content style="fixed-case">A</jats:styled-content> synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t<jats:sup>6</jats:sup><jats:styled-content style="fixed-case">A</jats:styled-content> is dispensable in <jats:italic>Deinococcus radiodurans</jats:italic>, <jats:italic>Thermus thermophilus</jats:italic>, <jats:italic>Synechocystis</jats:italic> <jats:styled-content style="fixed-case">PCC</jats:styled-content>6803 and <jats:styled-content style="fixed-case"><jats:italic>S</jats:italic></jats:styled-content><jats:italic>treptococcus mutans</jats:italic>. Proteomic analysis of t<jats:sup>6</jats:sup><jats:styled-content style="fixed-case">A</jats:styled-content><jats:sup>−</jats:sup> <jats:styled-content style="fixed-case"><jats:italic>D</jats:italic></jats:styled-content><jats:italic>. radiodurans</jats:italic> strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t<jats:sup>6</jats:sup><jats:styled-content style="fixed-case">A</jats:styled-content> in t<jats:styled-content style="fixed-case">RNA</jats:styled-content>s. Thus, although t<jats:sup>6</jats:sup><jats:styled-content style="fixed-case">A</jats:styled-content> is universally conserved in tRNAs, its role in translation might vary greatly between organisms.