The Doc-Phd toxin-antitoxin system inhibits bacterial translation via an unknown mechanism. Functional and structural analyses now show that Doc, which has an active site like AMPylating Fic proteins, actually works as a kinase, phosphorylating EF-Tu to block translation. Fic proteins are ubiquitous in all of the domains of life and have 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 having 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 EF-Tu 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 the catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities.

Stopping Transcription: It is as important to terminate any biological process as it is to start it. Transcription, copying information encoded in genes into RNA, requires accurate and timely termination. Nielsen et al. (p. 1577 ) present a mechanism for transcription termination by RNA polymerase III, the enzyme that synthesizes the majority of RNA molecules in eukaryotes. In this scenario, the folding of the RNA as it is transcribed by polymerase into a highly structured transcript causes termination at the end of its synthesis. This mechanism may serve as a control of proper folding of structural or catalytic RNAs synthesized by RNA polymerase III. Comparison with other organisms suggests that this mechanism emerged before divergence of bacteria and eukaryotes.

A combined structural, functional, and genetic approach was used to investigate inhibition of bacterial RNA polymerase (RNAP) by sorangicin (Sor), a macrolide polyether antibiotic. Sor lacks chemical and structural similarity to the ansamycin rifampicin (Rif), an RNAP inhibitor widely used to treat tuberculosis. Nevertheless, structural analysis revealed Sor binds in the same RNAP β subunit pocket as Rif, with almost complete overlap of RNAP binding determinants, and functional analysis revealed that both antibiotics inhibit transcription by directly blocking the path of the elongating transcript at a length of 2–3 nucleotides. Genetic analysis indicates that Rif binding is extremely sensitive to mutations expected to change the shape of the antibiotic binding pocket, while Sor is not. We suggest that conformational flexibility of Sor, in contrast to the rigid conformation of Rif, allows Sor to adapt to changes in the binding pocket. This has important implications for drug design against rapidly mutating targets.