Promiscuous behaviour of archaeal ribosomal proteins: implications for eukaryotic ribosome evolution.

In all living cells, protein synthesis occurs on ribonucleoprotein particles called ribosomes. Molecular models have been reported for complete bacterial 70S and eukaryotic 80S ribosomes; however, only molecular models of large 50S subunits have been reported for archaea. Here, we present a complete molecular model for the Pyrococcus furiosus 70S ribosome based on a 6.6 Å cryo-electron microscopy map. Moreover, we have determined cryo-electron microscopy reconstructions of the Euryarchaeota Methanococcus igneus and Thermococcus kodakaraensis 70S ribosomes and Crenarchaeota Staphylothermus marinus 50S subunit. Examination of these structures reveals a surprising promiscuous behavior of archaeal ribosomal proteins: We observe intersubunit promiscuity of S24e and L8e (L7ae), the latter binding to the head of the small subunit, analogous to S12e in eukaryotes. Moreover, L8e and L14e exhibit intrasubunit promiscuity, being present in two copies per archaeal 50S subunit, with the additional binding site of L14e analogous to the related eukaryotic r-protein L27e. Collectively, these findings suggest insights into the evolution of eukaryotic ribosomal proteins through increased copy number and binding site promiscuity.

Insights into the mode of action of novel fluoroketolides, potent inhibitors of bacterial protein synthesis.

Ketolides, the third generation of expanded-spectrum macrolides, have in the last years become a successful weapon in the endless war against macrolide-resistant pathogens. Ketolides are semisynthetic derivatives of the naturally produced macrolide erythromycin, displaying not only improved activity against some erythromycin-resistant strains but also increased bactericidal activity as well as inhibitory effects at lower drug concentrations. In this study, we present a series of novel ketolides carrying alkyl-aryl side chains at the C-6 position of the lactone ring and, additionally, one or two fluorine atoms attached either directly to the lactone ring at the C-2 position or indirectly via the C-13 position. According to our genetic and biochemical studies, these novel ketolides occupy the known macrolide binding site at the entrance of the ribosomal tunnel and exhibit lower MIC values against wild-type or mutant strains than erythromycin. In most cases, the ketolides display activities comparable to or better than the clinically used ketolide telithromycin. Chemical protection experiments using Escherichia coli ribosomes bearing U2609C or U754A mutations in 23S rRNA suggest that the alkyl-aryl side chain establishes an interaction with the U2609-A752 base pair, analogous to that observed with telithromycin but unlike the interactions formed by cethromycin. These findings reemphasize the versatility of the alkyl-aryl side chains with respect to species specificity, which will be important for future design of improved antimicrobial agents.

Localization of eukaryote-specific ribosomal proteins in a 5.5-Å cryo-EM map of the 80S eukaryotic ribosome.

Protein synthesis in all living organisms occurs on ribonucleoprotein particles, called ribosomes. Despite the universality of this process, eukaryotic ribosomes are significantly larger in size than their bacterial counterparts due in part to the presence of 80 r proteins rather than 54 in bacteria. Using cryoelectron microscopy reconstructions of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome, we have localized and modeled 74/80 (92.5%) of the ribosomal proteins, encompassing 12 archaeal/eukaryote-specific small subunit proteins as well as the complete complement of the ribosomal proteins of the eukaryotic large subunit. Near-complete atomic models of the 80S ribosome provide insights into the structure, function, and evolution of the eukaryotic translational apparatus.

Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-A resolution.

Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Šresolution. This map, together with a 6.1-Šmap of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES-ES and r-protein-ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.

PSRP1 is not a ribosomal protein, but a ribosome-binding factor that is recycled by the ribosome-recycling factor (RRF) and elongation factor G (EF-G).

Plastid-specific ribosomal proteins (PSRPs) have been proposed to play roles in the light-dependent regulation of chloroplast translation. Here we demonstrate that PSRP1 is not a bona fide ribosomal protein, but rather a functional homologue of the Escherichia coli cold-shock protein pY. Three-dimensional Cryo-electron microscopic (Cryo-EM) reconstructions reveal that, like pY, PSRP1 binds within the intersubunit space of the 70S ribosome, at a site overlapping the positions of mRNA and A- and P-site tRNAs. PSRP1 induces conformational changes within ribosomal components that comprise several intersubunit bridges, including bridge B2a, thereby stabilizes the ribosome against dissociation. We find that the presence of PSRP1/pY lowers the binding of tRNA to the ribosome. Furthermore, similarly to tRNAs, PSRP1/pY is recycled from the ribosome by the concerted action of the ribosome-recycling factor (RRF) and elongation factor G (EF-G). These results suggest a novel function for EF-G and RRF in the post-stress return of PSRP1/pY-inactivated ribosomes to the actively translating pool.

Shine-Dalgarno interaction prevents incorporation of noncognate amino acids at the codon following the AUG.

During translation, usually only one in approximately 400 misincorporations affects the function of a nascent protein, because only chemically similar near-cognate amino acids are misincorporated in place of the cognate one. The deleterious misincorporation of a chemically dissimilar noncognate amino acid during the selection process is precluded by the presence of a tRNA at the ribosomal E-site. However, the selection of first aminoacyl-tRNA, directly after initiation, occurs without an occupied E-site, i.e., when only the P-site is filled with the initiator tRNA and thus should be highly error-prone. Here, we show how bacterial ribosomes have solved this accuracy problem: In the absence of a Shine-Dalgarno (SD) sequence, the first decoding step at the A-site after initiation is extremely error-prone, even resulting in the significant incorporation of noncognate amino acids. In contrast, when a SD sequence is present, the incorporation of noncognate amino acids is not observed. This is precisely the effect that the presence of a cognate tRNA at the E-site has during the elongation phase. These findings suggest that during the initiation phase, the SD interaction functionally compensates for the lack of codon-anticodon interaction at the E-site by reducing the misincorporation of near-cognate amino acids and prevents noncognate misincorporation.

Non-hydrolyzable RNA-peptide conjugates: a powerful advance in the synthesis of mimics for 3'-peptidyl tRNA termini.

Translation of specific small peptides on the ribosome can confer resistance to macrolide antibiotics. To reveal the molecular details of this and related phenomena, stable RNA-peptide conjugates that mimic peptidyl-tRNA would be desirable, especially for ribosome structural biology. A flexible solid-phase synthesis strategy now allows efficient access to these highly requested derivatives without restriction on the RNA and peptide sequences.

Differential effects of thiopeptide and orthosomycin antibiotics on translational GTPases.

The ribosome is a major target in the bacterial cell for antibiotics. Here, we dissect the effects that the thiopeptide antibiotics thiostrepton (ThS) and micrococcin (MiC) as well as the orthosomycin antibiotic evernimicin (Evn) have on translational GTPases. We demonstrate that, like ThS, MiC is a translocation inhibitor, and that the activation by MiC of the ribosome-dependent GTPase activity of EF-G is dependent on the presence of the ribosomal proteins L7/L12 as well as the G' subdomain of EF-G. In contrast, Evn does not inhibit translocation but is a potent inhibitor of back-translocation as well as IF2-dependent 70S-initiation complex formation. Collectively, these results shed insight not only into fundamental aspects of translation but also into the unappreciated specificities of these classes of translational inhibitors.

Proteomic characterization of archaeal ribosomes reveals the presence of novel archaeal-specific ribosomal proteins.

Protein synthesis occurs in macromolecular particles called ribosomes. All ribosomes are composed of RNA and proteins. While the protein composition of bacterial and eukaryotic ribosomes has been well-characterized, a systematic analysis of archaeal ribosomes has been lacking. Here we report the first comprehensive two-dimensional PAGE and mass spectrometry analysis of archaeal ribosomes isolated from the thermophilic Pyrobaculum aerophilum and the thermoacidophilic Sulfolobus acidocaldarius Crenarchaeota. Our analysis identified all 66 ribosomal proteins (r-proteins) of the P. aerophilum small and large subunits, as well as all but two (62 of 64; 97%) r-proteins of the S. acidocaldarius small and large subunits that are predicted genomically. Some r-proteins were identified with one or two lysine methylations and N-terminal acetylations. In addition, we identify three hypothetical proteins that appear to be bona fide r-proteins of the S. acidocaldarius large subunit. Dissociation of r-proteins from the S. acidocaldarius large subunit indicates that the novel r-proteins establish tighter interactions with the large subunit than some integral r-proteins. Furthermore, cryo electron microscopy reconstructions of the S. acidocaldarius and P. aerophilum 50S subunits allow for a tentative localization of the binding site of the novel r-proteins. This study illustrates not only the potential diversity of the archaeal ribosomes but also the necessity to experimentally analyze the archaeal ribosomes to ascertain their protein composition. The discovery of novel archaeal r-proteins and factors may be the first step to understanding how archaeal ribosomes cope with extreme environmental conditions.

Maintaining the ribosomal reading frame: the influence of the E site during translational regulation of release factor 2.

Maintenance of the translation reading frame is one of the most remarkable achievements of the ribosome while decoding the information of an mRNA. Loss of the reading frame through spontaneous frameshifting occurs with a frequency of one in 30,000 amino acid incorporations. However, at many recoding sites, the mechanism that controls reading frame maintenance is switched off. One such example is the programmed +1 frameshift site of the prfB gene encoding the termination factor RF2, in which slippage into the forward frame by one nucleotide can attain an efficiency of approximately 100%, namely, four orders of magnitude higher than normally observed. Here, using the RF2 frameshift window, we demonstrate that premature release of the E site tRNA from the ribosome is coupled with high-level frameshifting. Consistently, in a minimal system, the presence of the E site tRNA prevents the +1 frameshift event, illustrating the importance of the E site for reading-frame maintenance.