Iron-mediated degradation of ribosomes under oxidative stress is attenuated by manganese

Protein biosynthesis is fundamental to cellular life and requires the efficient functioning of the translational machinery. At the center of this machinery is the ribosome, a ribonucleoprotein complex that depends heavily on Mg²⁺ for structure. Recent work has indicated that other metal cations can substitute for Mg²⁺, raising questions about the role different metals may play in the maintenance of the ribosome under oxidative stress conditions. Here, we assess ribosomal integrity following oxidative stress both in vitro and in cells to elucidate details of the interactions between Fe²⁺ and the ribosome and identify Mn²⁺ as a factor capable of attenuating oxidant-induced Fe²⁺-mediated degradation of rRNA. We report that Fe²⁺ promotes degradation of all rRNA species of the yeast ribosome and that it is bound directly to RNA molecules. Furthermore, we demonstrate that Mn²⁺ competes with Fe²⁺ for rRNA-binding sites and that protection of ribosomes from Fe²⁺-mediated rRNA hydrolysis correlates with the restoration of cell viability. Our data, therefore, suggest a relationship between these two transition metals in controlling ribosome stability under oxidative stress.     

Reduced ribosomes of the apicoplast and mitochondrion of Plasmodium spp. and predicted interactions with antibiotics

Apicomplexan protists such as Plasmodium and Toxoplasma contain a mitochondrion and a relic plastid (apicoplast) that are sites of protein translation. Although there is emerging interest in the partitioning and function of translation factors that participate in apicoplast and mitochondrial peptide synthesis, the composition of organellar ribosomes remains to be elucidated. We carried out an analysis of the complement of core ribosomal protein subunits that are encoded by either the parasite organellar or nuclear genomes, accompanied by a survey of ribosome assembly factors for the apicoplast and mitochondrion. A cross-species comparison with other apicomplexan, algal and diatom species revealed compositional differences in apicomplexan organelle ribosomes and identified considerable reduction and divergence with ribosomes of bacteria or characterized organelle ribosomes from other organisms. We assembled structural models of sections of Plasmodium falciparum organellar ribosomes and predicted interactions with translation inhibitory antibiotics. Differences in predicted drug–ribosome interactions with some of the modelled structures suggested specificity of inhibition between the apicoplast and mitochondrion. Our results indicate that Plasmodium and Toxoplasma organellar ribosomes have a unique composition, resulting from the loss of several large and small subunit proteins accompanied by significant sequence and size divergences in parasite orthologues of ribosomal proteins.     

The slow dissociation rate of K-1602 contributes to the enhanced inhibitory activity of this novel alkyl–aryl-bearing fluoroketolide

Ketolides belong to the latest generation of macrolides and are not only effective against macrolide susceptible bacterial strains but also against some macrolide resistant strains. Here we present data providing insights into the mechanism of action of K-1602, a novel alkyl–aryl-bearing fluoroketolide. According to our data, the K-1602 interacts with the ribosome as a one-step slow binding inhibitor, displaying an association rate constant equal to 0.28?×?10^4?M^?1 s^?1 and a dissociation rate constant equal to 0.0025?min^?1. Both constants contribute to produce an overall inhibition constant K_i equal to 1.49?×?10^?8?M, which correlates very well with the superior activity of this compound when compared with many other ketolides or fluoroketolides.     

Ribosome profiles and riboproteomes of healthy and Potato virus A- and Agrobacterium-infected Nicotiana benthamiana plants

Nicotiana benthamiana is an important model plant for plant–microbe interaction studies. Here, we compared ribosome profiles and riboproteomes of healthy and infected N. benthamiana plants. We affinity purified ribosomes from transgenic leaves expressing a FLAG-tagged ribosomal large subunit protein RPL18B of Arabidopsis thaliana. Purifications were prepared from healthy plants and plants that had been infiltrated with Agrobacterium tumefaciens carrying infectious cDNA of Potato virus A (PVA) or firefly luciferase gene, referred to here as PVA- or Agrobacterium-infected plants, respectively. Plants encode a number of paralogous ribosomal proteins (r-proteins). The N. benthamiana riboproteome revealed approximately 6600 r-protein hits representing 424 distinct r-proteins that were members of 71 of the expected 81 r-protein families. Data are available via ProteomeXchange with identifier PXD011602. The data indicated that N. benthamiana ribosomes are heterogeneous in their r-protein composition. In PVA-infected plants, the number of identified r-protein paralogues was lower than in Agrobacterium-infected or healthy plants. A. tumefaciens proteins did not associate with ribosomes, whereas ribosomes from PVA-infected plants co-purified with viral cylindrical inclusion protein and helper component proteinase, reinforcing their possible role in protein synthesis during virus infection. In addition, viral NIa protease-VPg, RNA polymerase NIb and coat protein were occasionally detected. Infection did not affect the proportions of ribosomal subunits or the monosome to polysome ratio, suggesting that no overall alteration in translational activity took place on infection with these pathogens. The riboproteomic data of healthy and pathogen-infected N. benthamiana will be useful for studies on the specific use of r-protein paralogues to control translation in infected plants.     

Correlation of the structure and conformational changes of selected fragments of plant small ribosomal RNA within the steps of polypeptide chain elongation

The interaction and conformational relationships between rRNAs and ribosomal proteins are responsible for ribosome activity. We tested seven different deoxyoligonucleotides complementary to the selected, highly conserved sequences of 18S rRNAs important in protein biosynthesis. We carried out a reaction of binding Phe-tRNA to A site on the ribosomes converted either to pre- or to post-translocational states (with or without pre-hybridized oligonucleotides). We found a correlation between the level of oligomer hybridization and the inhibition of AA-tRNA binding. We observed well-defined structural changes of ribosome's conformation during different steps of the elongation cycle of protein biosynthesis.     

Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo‐tomography

We employed electron cryo‐tomography to visualize cytosolic ribosomes on the surface of mitochondria. Translation‐arrested ribosomes reveal the clustered organization of the TOM complex, corroborating earlier reports of localized translation. Ribosomes are shown to interact specifically with the TOM complex, and nascent chain binding is crucial for ribosome recruitment and stabilization. Ribosomes are bound to the membrane in discrete clusters, often in the vicinity of the crista junctions. This interaction highlights how protein synthesis may be coupled with transport. Our work provides unique insights into the spatial organization of cytosolic ribosomes on mitochondria.     

The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome

RelE/RelB is a well-characterized toxin-anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. RelE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes also in eukaryotes. RelE cleavage in the A-site codon of a stalled eukaryote ribosome is precise and easily monitored, making "RelE printing" a useful complement to toeprinting to determine the exact mRNA location on the eukaryote ribosome and to probe the occupancy of its A site.     

Cytokinins and cytokinin-binding sites in transgenic potato plants

The introduction of the gene for cytokinin biosynthesis into the potato genome led to a manifold increase in the level of cytokinins (zeatin and zeatin riboside) in transgenic plants grown in vitro. The high amount of endogenous cytokinins in 20-day-old plants of clone 1339-3A correlated with high cytokinin-binding capacity of ribosomes that is presumably attribute to a cytokinin receptor.     

Functional 70S hybrid ribosomes from blue-green algae and bacteria

Hybrid 70S ribosomes were produced by combining Anacystis nidulans and Escherichia coli 30S and 50S subunits. Both the A. nidulans 30S-E. coli 50S and E. coli 30S- A. nidulans 50S hybrids were functional in synthesizing protein when tested in a standard in vitro amino acid incorporating system. Both 70S hybrids were inhibited by streptomycin but the degree of inhibition was dependent upon the source of the 30S subunit. The ability to form functional 70S ribosomes from subunits of blue-green algae and bacteria is further evidence of the procaryotic nature of blue-greens and of the functional homology of the two protein synthesizing systems.