HipA of Escherichia coli is a eukaryote-like serine-threonine kinase that inhibits cell growth and induces persistence (multidrug tolerance). Previously, it was proposed that HipA inhibits cell growth by the phosphorylation of the essential translation factor EF-Tu. Here, we provide evidence that EF-Tu is not a target of HipA. Instead, a genetic screen reveals that the overexpression of glutamyl-tRNA synthetase (GltX) suppresses the toxicity of HipA. We show that HipA phosphorylates conserved Ser(239) near the active center of GltX and inhibits aminoacylation, a unique example of an aminoacyl-tRNA synthetase being inhibited by a toxin encoded by a toxin-antitoxin locus. HipA only phosphorylates tRNA(Glu)-bound GltX, which is consistent with the earlier finding that the regulatory motif containing Ser(239) changes configuration upon tRNA binding. These results indicate that HipA mediates persistence by the generation of "hungry" codons at the ribosomal A site that trigger the synthesis of (p)ppGpp, a hypothesis that we verify experimentally.

Fic proteins are ubiquitous in all domains of life and play critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc/phd toxin/antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to the predicted AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering it unable to bind aminoacylated tRNAs. We provide evidence that EF-Tu phosphorylation diverged from AMPylation by antiparallel binding of the NTP relative to the catalytic residues of the conserved Fic catalytic core of Doc. The results bring insights into the mechanism and role of phosphorylation of EF-Tu in bacterial physiology as well as represent an example of catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities.

In bacteria, the first two steps of gene expression—transcription and translation—are spatially and temporally coupled. Uncoupling may lead to the arrest of transcription through RNA polymerase backtracking, which interferes with replication forks, leading to DNA double-stranded breaks and genomic instability. How transcription–translation coupling mitigates these conflicts is unknown. Here we show that, unlike replication, translation is not inhibited by arrested transcription elongation complexes. Instead, the translating ribosome actively pushes RNA polymerase out of the backtracked state, thereby reactivating transcription. We show that the distance between the two machineries upon their contact on mRNA is smaller than previously thought, suggesting intimate interactions between them. However, this does not lead to the formation of a stable functional complex between the enzymes, as was once proposed. Our results reveal an active, energy-driven mechanism that reactivates backtracked elongation complexes and thus helps suppress their interference with replication.

Transcription and translation are coupled in bacteria, meaning that translation takes place co-transcriptionally. During transcription–translation, both machineries mutually affect each others’ functions, which is important for regulation of gene expression. Analysis of interactions between RNA polymerase (RNAP) and the ribosome, however, are limited due to the lack of an in vitro experimental system. Here, we report the development of an in vitro transcription coupled to translation system assembled from purified components. The system allows controlled stepwise transcription and simultaneous stepwise translation of the nascent RNA, and permits investigation of the interactions of RNAP with the ribosome, as well as the effects of translation on transcription and transcription on translation. As an example of usage of this experimental system, we uncover complex effects of transcription–translation coupling on pausing of transcription.

Phosphorylation-induced conformational trap is an essential mechanism for phosphoregulation of bacterial metabolism.