Background: Toxin-antitoxin (TA) systems play a crucial role in bacterial survival during stress. Results: Structures of the P. vulgaris HigBA complex reveal novel structural features such as the HigB and HigA interaction and the solvent accessibility of the HigB active site. Conclusion: Antitoxin HigA interacts with toxin HigB in a novel manner. Significance: Our results emphasize that antitoxins are a structurally diverse class of proteins.

Maintenance of the correct reading frame on the ribosome is essential for accurate protein synthesis. Here, we report structures of the 70S ribosome bound to frameshift suppressor tRNA SufA6 and N1-methylguanosine at position 37 (m 1 G37) modification-deficient anticodon stem loop Pro , both of which cause the ribosome to decode 4 rather than 3 nucleotides, resulting in a +1 reading frame. Our results reveal that decoding at +1 suppressible codons causes suppressor tRNA SufA6 to undergo a rearrangement of its 5′ stem that destabilizes U32, thereby disrupting the conserved U32-A38 base pair. Unexpectedly, the removal of the m 1 G37 modification of tRNA Pro also disrupts U32-A38 pairing in a structurally analogous manner. The lack of U32-A38 pairing provides a structural correlation between the transition from canonical translation and a +1 reading of the mRNA. Our structures clarify the molecular mechanism behind suppressor tRNA-induced +1 frameshifting and advance our understanding of the role played by the ribosome in maintaining the correct translational reading frame.near cognate | processivity T he three polymerase reactions of DNA replication, RNA transcription, and protein translation are essential to life and all involve a delicate balance between speed and fidelity. The bacterial ribosome decodes three mRNA nucleotides into a single amino acid at a rate of ∼20 residues/s with high fidelity (10 3

Background: Toxin-antitoxin complexes autoregulate transcription depending upon growth conditions. Results: DinJ-YafQ structure was determined, and minimal requirements for transcriptional autorepression were identified. Conclusion:The dinJyafQ operon is regulated in a novel manner by either DinJ-YafQ-or LexA-mediated repression. Significance: Our results reveal new mechanistic insights into the action of DinJ-YafQ as a transcriptional repressor.

Bacteria contain multiple type II toxins that selectively degrade mRNAs bound to the ribosome to regulate translation and growth and facilitate survival during the stringent response. Ribosomedependent toxins recognize a variety of three-nucleotide codons within the aminoacyl (A) site, but how these endonucleases achieve substrate specificity remains poorly understood. Here, we identify the critical features for how the host inhibition of growth B (HigB) toxin recognizes each of the three A-site nucleotides for cleavage. X-ray crystal structures of HigB bound to two different codons on the ribosome illustrate how HigB uses a microbial RNase-like nucleotide recognition loop to recognize either cytosine or adenosine at the second A-site position. Strikingly, a single HigB residue and 16S rRNA residue C1054 form an adenosine-specific pocket at the third A-site nucleotide, in contrast to how tRNAs decode mRNA. Our results demonstrate that the most important determinant for mRNA cleavage by ribosome-dependent toxins is interaction with the third A-site nucleotide. (1,3,4). This rapid inhibitory switch suppresses metabolite consumption and temporarily halts cell growth to promote bacterial survival until nutrients are readily available. Among the prosurvival genes regulated by (p)ppGpp production are toxin-antitoxin modules, which have additional roles in antibiotic resistance and tolerance, biofilm and persister cell formation, and niche-specific colonization (5-11). The critical roles toxin-antitoxin pairs play in bacterial physiology underscore the importance of understanding their molecular targets and modes of action.There are five different classes (I to V) of toxin-antitoxin systems defined by how the antitoxin represses toxin function (1). Type II toxin-antitoxin pairs form protein-protein complexes during exponential growth that serve two purposes: inhibition of toxin activity by antitoxin binding and transcriptional autorepression to limit toxin expression (12). Antitoxins are proteolytically degraded after (p)ppGpp accumulation, leading to derepression at the toxin-antitoxin promoter (8, 12). Liberated toxin proteins inhibit the replication or translation machinery by targeting DNA gyrase, initiator tRNA fMet , glutamyl-tRNA synthetase, EF-Tu, free mRNA, ribosome-bound mRNA, and the ribosome itself (13-20).Ribosome-dependent toxins cleave mRNAs on the ribosome between the second and third nucleotides of the aminoacyl (A)-site codon (21-23). Although collectively Escherichia coli ribosome-dependent toxins target a diverse range of codons, each individual toxin appears to have a strong codon preference and cleaves at defined positions along the mRNA (24-26). RelE cleaves at UAG stop codons and the CAG sense codon (all codons denoted in the 5′-3′ direction); YoeB cleaves at codons following a translational AUG start site and at the UAA stop codon; and YafQ cleaves a single AAA sense codon (16,24,(27)(28)(29). In contrast, Proteus vulgaris host inhibition of growth B (HigB) toxin degrades multiple codons encod...

After four decades of research aimed at understanding tRNA selection on the ribosome, the mechanism by which ribosomal ambiguity (ram) mutations promote miscoding remains unclear. Here, we present two X-ray crystal structures of the Thermus thermophilus 70S ribosome containing 16S rRNA ram mutations, G347U and G299A. Each of these mutations causes miscoding in vivo and stimulates elongation factor thermo unstable (EF-Tu)-dependent GTP hydrolysis in vitro. Mutation G299A is located near the interface of ribosomal proteins S4 and S5 on the solvent side of the subunit, whereas G347U is located 77 Å distant, at intersubunit bridge B8, close to where EF-Tu engages the ribosome. Despite these disparate locations, both mutations induce almost identical structural rearrangements that disrupt the B8 bridge-namely, the interaction of h8/h14 with L14 and L19. This conformation most closely resembles that seen upon EF-Tu·GTP·aminoacyl-tRNA binding to the 70S ribosome. These data provide evidence that disruption and/or distortion of B8 is an important aspect of GTPase activation. We propose that, by destabilizing B8, G299A and G347U reduce the energetic cost of attaining the GTPase-activated state and thereby decrease the stringency of decoding. This previously unappreciated role for B8 in controlling the decoding process may hold relevance for many other ribosomal mutations known to influence translational fidelity.T he molecular mechanisms controlling the fidelity of DNA replication, transcription, and translation have been areas of intense interest since the discovery of the genetic code. Thermodynamic differences between standard Watson-Crick and alternative (e.g., wobble) base pairs in solution are insufficient to explain the high fidelity for any of the three polymerase reactions of the central dogma (1), indicating an active role for the enzymes in substrate selectivity (1-3). Mechanistic studies of polymerases have revealed some common themes, such as the specific recognition of Watson-Crick base pair geometry, larger forward rate constants for correct substrates (induced fit), separate opportunities for incorrect substrate rejection (kinetic proofreading), and postincorporation correction mechanisms (1-5).During translation, the ribosome must select aminoacyl-tRNA (aa-tRNA) substrates based on the mRNA sequence. Extensive biochemical studies have shed light on the kinetics of this decoding process (reviewed in ref. 6). The aa-tRNA is delivered to the ribosome as part of a ternary complex (TC) with elongation factor thermo unstable (EF-Tu) and GTP. Initial binding of TC, mediated primarily by L7/L12 of the 50S subunit, is followed by the sampling of codon-anticodon interactions in the 30S A site. Codon-anticodon pairing leads to GTPase activation and GTP hydrolysis, which allows release of the acceptor end of aa-tRNA from EF-Tu. The aa-tRNA then either moves completely into the ribosomal A site (a step termed accommodation), where it can participate in peptide bond formation, or is rejected and released into solution...