Thiostrepton inhibits the turnover but not the GTPase of elongation factor G on the ribosome

The region around position 1067 in domain II of 23S rRNA frequently is referred to as the GTPase center of the ribosome. The notion is based on the observation that the binding of the antibiotic thiostrepton to this region inhibited GTP hydrolysis by elongation factor G (EF-G) on the ribosome at the conditions of multiple turnover. In the present work, we have reanalyzed the mechanism of action of thiostrepton. Results obtained by biochemical and fast kinetic techniques show that thiostrepton binding to the ribosome does not interfere with factor binding or with single-round GTP hydrolysis. Rather, the antibiotic inhibits the function of EF-G in subsequent steps, including release of inorganic phosphate from EF-G after GTP hydrolysis, tRNA translocation, and the dissociation of the factor from the ribosome, thereby inhibiting the turnover reaction. Structurally, thiostrepton interferes with EF-G footprints in the α-sarcin stem loop (A2660, A2662) located in domain VI of 23S rRNA. The results indicate that thiostrepton inhibits a structural transition of the 1067 region of 23S rRNA that is important for functions of EF-G after GTP hydrolysis

Purine bases at position 37 of tRNA stabilize codon–anticodon interaction in the ribosomal A site by stacking and Mg(2+)-dependent interactions

The anticodon loop of tRNA contains a number of conserved or semiconserved nucleotides. In most tRNAs, a highly modified purine is found at position 37 immediately 3′ to the anticodon. Here, we examined the role of the base at position 37 for tRNA(Phe) binding to the A site of Escherichia coli ribosomes. Affinities and rate constants of A-site binding of native yeast peptidyl-tRNA(Phe) with hypermodified G (wybutine), or of unmodified peptidyl-tRNA(Phe) transcripts with G, A, C, or U, at position 37 were measured. The data indicate that purines stabilize binding due to stronger stacking and additional interactions with the ribosome mediated by Mg(2+) ions. Paromomycin, an antibiotic that binds to 16S rRNA in the decoding center, greatly stabilized tRNAs in the A site and abolished the Mg(2+)-dependence of binding. Comparison of binding enthalpies and entropies suggests that hypermodification of the base at position 37 does not affect stacking in the codon–anticodon complex, but rather decreases the entropic penalty for A-site binding. Substitution of purines with pyrimidines at position 37 increases the rates of tRNA binding to and dissociation from the A site. The data suggest that initial binding of tRNA to the A site is followed by a rate-limiting rearrangement of the anticodon loop or the ribosome decoding center that is favored by purines at position 37 and involves stronger stacking, additional Mg(2+) binding, and interactions with 16S rRNA

Exploration of the conserved A+C wobble pair within the ribosomal peptidyl transferase center using affinity purified mutant ribosomes

Protein synthesis in the ribosome's large subunit occurs within an active site comprised exclusively of RNA. Mutational studies of rRNA active site residues could provide valuable insight into the mechanism of peptide bond formation, but many of these mutations cause a dominant lethal phenotype, which prevents production of the homogeneous mutant ribosomes needed for analysis. We report a general method to affinity purify in vivo assembled 50S ribosomal subunits containing lethal active site mutations via a U1A protein-binding tag inserted onto the 23S rRNA. The expected pH-dependent formation of the A2450+C2063 wobble pair has made it a potential candidate for the pH-dependent conformational change that occurs within the ribosomal active site. Using this approach, the active site A2450+C2063 pair was mutated to the isosteric, but pH-independent, G2450•U2063 wobble pair, and 50S subunits containing the mutations were affinity purified. The G•U mutation caused the adjacent A2451 to become hyper-reactive to dimethylsulfate (DMS) modification in a pH-independent manner. Furthermore, the G•U mutation decreased both the rate of peptide bond formation and the affinity of the post-translocation complex for puromycin. The reaction rate (k(pep)) was reduced ∼200-fold for both puromycin and the natural aminoacyl-tRNA A-site substrate. The mutations also substantially altered the pH dependence of the reaction. Mutation of this base pair has significant deleterious effects upon peptidyl transferase activity, but because G•U mutation disrupts several tertiary contacts with the wobble pair, the assignment of A2450 as the active site residue with the neutral pK(a) important for the peptidyl transferase reaction cannot be fully supported or excluded based upon these data

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P < 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)