mRNA bound to the 30S subunit is a HigB toxin substrate

Abstract

Activation of bacterial toxins during stress results in cleavage of mRNAs in the context of the ribosome. These toxins are thought to function as global translational inhibitors yet recent studies suggest each may have distinct mRNA specificities that result in selective translation for bacterial survival. Here we demonstrate that mRNA in the context of a bacterial 30S subunit is sufficient for ribosome-dependent toxin HigB endonucleolytic activity, suggesting that HigB interferes with the initiation step of translation. We determined the X-ray crystal structure of HigB bound to the 30S, revealing that two solvent-exposed clusters of HigB basic residues directly interact with 30S 16S rRNA helices 18, 30, and 31. We further show that these HigB residues are essential for ribosome recognition and function. Comparison with other ribosome-dependent toxins RelE and YoeB reveals that each interacts with similar features of the 30S aminoacyl (A) site yet does so through presentation of diverse structural motifs.

Alternative Conformations and Motions Adopted by 30S Ribosomal Subunits Visualized by Cryo-Electron Microscopy

“…Surprisingly, we also found the density representing uS2 to be completely absent from our cryo-EM maps. In the 30S subunit, uS2 binds in the solvent face (convex face) of the subunit stably anchoring its two domains to the 16S rRNA and typically is fully visible in previously obtained X-ray (Schureck et al, 2016;Wimberly et al, 2000) and cryo-EM In cryo-EM, to preserve the specimen in their hydrated state, one spread the sample in a thin layer of buffer solution supported in the cryo-EM grid right before plunging the grid into liquid ethane to freeze the liquid layer into vitreous ice. In the vitrification device used in our experiments, the time that elapses between the blotting of the grid to form the thin layer and the vitrification is typically 1 second.…”

The Variety in the Common Theme of Translation Inhibition by Type II Toxin–Antitoxin Systems

“…The YafO toxin was shown to be a ribosome-dependent endoribonuclease that cleaves mRNAs 11-13 bases downstream of the initiation codon (Zhang et al, 2009;Christensen-Dalsgaard et al, 2010). Such cleavage event was speculated to be located near the mRNA entrance tunnel rather than at the A site as determined for the RelE-like toxins (Schureck et al, 2016a). Although initially YafO was considered a RelE-like toxin (Christensen-Dalsgaard et al, 2010), the lack of structural information about this toxin or its interaction with ribosome lead to YafO being classified as a separate family (Leplae et al, 2011).…”

“…RelE Nsp , Sinorhizobium meliloti RelE Sme and Treponema denticola RelE Tde (Prysak et al, 2009;Goeders et al, 2013). P. vulgaris HigB cleaves A-rich codons (Hurley and Woychik, 2009;Schureck et al, 2016b), and was shown to cleave the 30S bound mRNA, indicating that it can interfere with the initiation step of translation after IF1 dissociation (Schureck et al, 2016a). The AAA and other A-rich codons are the most frequent at the beginning of the ORFs in E. coli (Sato et al, 2001).…”

“…It remains unclear why toxins rely on the ribosome for activity as simply blocking the translation cleavage of free mRNA would both be sufficient and efficient. It has been suggested that this dependence may indicate a specialized mechanism that would allow a response to stresses (Schureck et al, 2016a). On the other hand, selectivity for specific ribosomal complexes, such as initiating ribosomes, might be an efficient way to inhibit translation and is likely to give the same effect of reducing the global translation rate as ribosome-independent but more sequence-specific mRNA cleavage.…”

Translation‐dependent mRNA cleavage by YhaV in Escherichia coli

“…A HigB homolog encoded in the Proteus vulgaris Rts1 plasmid associates with the 50S ribosomal subunit in polysome profiling assay and it cleaves mRNA at AAA sequences and other cleavage sites regardless of the frame [16]. In contrast, according to the crystal structure, HigB also binds to the 30S ribosomal subunit [20].…”