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Chromosomally-encoded toxin-antitoxin complexes are ubiquitous in bacteria and regulate growth through the release of the toxin component typically in a stress-dependent manner. Type II ribosome-dependent toxins adopt a RelE-family RNase fold and inhibit translation by degrading mRNAs while bound to the ribosome. Here, we present biochemical and structural studies of the Escherichia coli YoeB toxin interacting with both a UAA stop and an AAU sense codon in pre- and post-mRNA cleavage states to provide insights into possible mRNA substrate selection. Both mRNAs undergo minimal changes during the cleavage event in contrast to type II ribosome-dependent RelE toxin. Further, the 16S rRNA decoding site nucleotides that monitor the mRNA in the aminoacyl(A) site adopt different orientations depending upon which toxin is present. Although YoeB is a RelE family member, it is the sole ribosome-dependent toxin that is dimeric. We show that engineered monomeric YoeB is active against mRNAs bound to both the small and large subunit. However, the stability of monomeric YoeB is reduced ∼20°C, consistent with potential YoeB activation during heat shock in E. coli as previously demonstrated. These data provide a molecular basis for the ability of YoeB to function in response to thermal stress., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Decoding is thought to be governed by a conformational transition in the ribosome-open (off) to closed (on)-that occurs upon codon-anticodon pairing in the A site. Ribosomal ambiguity (ram) mutations increase miscoding and map to disparate regions, consistent with a role for ribosome dynamics in decoding, yet precisely how these mutations act has been unclear. Here, we solved crystal structures of 70S ribosomes harboring 16S ram mutations G299A and G347U in the absence A-site tRNA (A-tRNA) and in the presence of a near-cognate anticodon stem-loop (ASL). In the absence of an A-tRNA, each of the mutant ribosomes exhibits a partially closed (on) state. In the 70S-G347U structure, the 30S shoulder is rotated inward and intersubunit bridge B8 is disrupted. In the 70S-G299A structure, the 30S shoulder is rotated inward and decoding nucleotide G530 flips into the anti conformation. Both of these mutant ribosomes adopt the fully closed (on) conformation in the presence of near-cognate A-tRNA, just as they do with cognate A-tRNA. Thus, these ram mutations act by promoting the open (off) to closed (on) transition, albeit in somewhat distinct ways. This work reveals the functional importance of 30S shoulder rotation for productive aminoacylated-tRNA incorporation., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacterial type II toxin-antitoxin modules are protein-protein complexes whose functions are finely tuned by rapidly changing environmental conditions. E. coli toxin YafQ is suppressed under steady state growth conditions by virtue of its interaction with its cognate antitoxin, DinJ. During stress, DinJ is proteolytically degraded and free YafQ halts translation by degrading ribosome-bound mRNA to slow growth until the stress has passed. Although structures of the ribosome with toxins RelE and YoeB have been solved, it is unclear what residues among ribosome-dependent toxins are essential for mediating both recognition of the ribosome and the mRNA substrate given their low sequence identities. Here we show that YafQ coordinates binding to the 70S ribosome via three surface-exposed patches of basic residues that we propose directly interact with 16S rRNA. We demonstrate that YafQ residues H50, H63, D67 and H87 participate in acid-base catalysis during mRNA hydrolysis and further show that H50 and H63 functionally complement as general bases to initiate the phosphodiester cleavage reaction. Moreover YafQ residue F91 likely plays an important role in mRNA positioning. In summary, our findings demonstrate the plasticity of ribosome-dependent toxin active site residues and further our understanding of which toxin residues are important for function., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Increasingly exact measurement of single crystal X-ray diffraction data offers detailed characterization of DNA conformation, hydration and electrostatics. However, instead of providing a more clear and unambiguous image of DNA, highly accurate diffraction data reveal polymorphism of the DNA atomic positions and conformation and hydration. Here we describe an accurate X-ray structure of B-DNA, painstakingly fit to a multistate model that contains multiple competing positions of most of the backbone and of entire base pairs. Two of ten base-pairs of CCAGGCCTGG are in multiple states distinguished primarily by differences in slide. Similarly, all the surrounding ions are seen to fractionally occupy discrete competing and overlapping sites. And finally, the vast majority of water molecules show strong evidence of multiple competing sites. Conventional resolution appears to give a false sense of homogeneity in conformation and interactions of DNA. In addition, conventional resolution yields an average structure that is not accurate, in that it is different from any of the multiple discrete structures observed at high resolution. Because base pair positional heterogeneity has not always been incorporated into model-building, even some high and ultrahigh-resolution structures of DNA do not indicate the full extent of conformational polymorphism.