The structures of antibiotics bound to the E site region of the 50 S ribosomal subunit of Haloarcula marismortui: 13-deoxytedanolide and girodazole

Crystal structures of the 50 S ribosomal subunit from Haloarcula marismortui complexed with two antibiotics have identified new sites at which antibiotics interact with the ribosome and inhibit protein synthesis. 13-Deoxytedanolide binds to the E site of the 50 S subunit at the same location as the CCA of tRNA, and thus appears to inhibit protein synthesis by competing with deacylated tRNAs for E site binding. Girodazole binds near the E site region, but is somewhat buried and may inhibit tRNA binding by interfering with conformational changes that occur at the E site. The specificity of 13-deoxytedanolide for eukaryotic ribosomes is explained by its extensive interactions with protein L44e, which is an E site component of archaeal and eukaryotic ribosomes, but not of eubacterial ribosomes. In addition, protein L28, which is unique to the eubacterial E site, overlaps the site occupied by 13-deoxytedanolide, precluding its binding to eubacterial ribosomes. Girodazole is specific for eukarytes and archaea because it makes interactions with L15 that are not possible in eubacteria.     

Functional interactions of an Escherichia coli ribosomal ATPase.

The gene encoding ribosome-bound ATPase (RbbA), which occurs bound to 70S ribosomes and 30S subunits, has been identified. The amino-acid sequence of RbbA reveals the presence of two ATP-binding domains in the N-terminal half of the protein. RbbA harbors an intrinsic ATPase activity that is stimulated by both 70S ribosomes and 30S subunits. Here we show that purified recombinant RbbA markedly stimulates polyphenylalanine synthesis in the presence of the elongation factors Tu and G (EF-Tu and EF-G) and that the hydrolysis of ATP by RbbA is required to stimulate synthesis. RbbA is reported to have affinity for EF-Tu but not for EF-G. Additionally, RbbA copurifies with 30S ribosomal subunits and can be crosslinked to the ribosomal protein S1. Studies using a spectrum of antibiotics, including some of similar function, revealed that hygromycin, which binds to the 30S subunit, has a significant effect on the ATPase activity and on the affinity of RbbA for ribosomes. A possible role for RbbA in protein-chain elongation is proposed.     

Fluorescence studies of the binding of a yeast tRNAPhe derivative to Escherichia coli ribosomes.

The equilibrium binding of a highly fluorescent derivative of yeast tRNA^Phe to Escherichia coli 70 S ribosomes was studied fluorimetrically at 7 °C in 25 mm-magnesium. Under these conditions 70 S ribosomes bind two deacylated tRNAs stoichiometrically. An analysis of the binding data using a model in which occupancy of the weaker site requires prior occupancy of the stronger site leads to apparent association constants of (1.00 ± 0.05) × 10⁹m^?1 and (3.4 ± 0.2) × 10⁷m^?1. The use of an independent site model does not change these values appreciably. The observed binding constants do not depend upon the presence or absence of the messenger RNA, poly(U). However, spectroscopic evidence strongly suggests that the anticodons of both bound tRNAs are in contact with the message. This evidence further suggests that in the presence of poly(U) the environment of the hypermodified base adjacent to the anticodon is substantially different in the two sites. This may reflect a difference in the conformation of the anticodon loops or an interaction between the hypermodified base of the weak site tRNA and the anticodon loop of the strong site tRNA.     

tmRNA·SmpB complex mimics native aminoacyl-tRNAs in the A site of stalled ribosomes

Bacterial ribosomes stalled on faulty, often truncated, mRNAs lacking stop codons are rescued by trans-translation. It relies on an RNA molecule (tmRNA) capable of replacing the faulty mRNA with its own open reading frame (ORF). Translation of tmRNA ORF results in the tagging of faulty protein for degradation and its release from the ribosome. We used single-particle cryo-electron microscopy to visualize tmRNA together with its helper protein SmpB on the 70S Escherichia coli ribosome in states subsequent to GTP hydrolysis on elongation factor Tu (EF-Tu). Three-dimensional reconstruction and heterogeneity analysis resulted in a 15 Å resolution structure of the tmRNA·SmpB complex accommodated in the A site of the ribosome, which shows that SmpB mimics the anticodon- and D-stem of native tRNAs missing in the tRNA-like domain of tmRNA. We conclude that the tmRNA·SmpB complex accommodates in the ribosomal A site very much like an aminoacyl-tRNA during protein elongation.     

The functional role of the C-terminal tail of the human ribosomal protein uS19

The eukaryotic ribosomal protein uS19 has a C-terminal tail that is absent in its bacterial homologue. This tail has been shown to be involved in the formation of the decoding site of human ribosomes. We studied here the previously unexplored functional significance of the 15 C-terminal amino acid residues of human uS19 for the assembly of ribosomes and translation using HEK293-based cell cultures capable of producing FLAG-labeled uS19 (uS19^FLAG) or its mutant form deprived of the mentioned amino acid ones. The examination of polysome profiles of cytoplasmic extracts from the respective cells revealed that the deletion of the above uS19 amino acid residues barely affected the assembly and maturation of 40S subunits and the initiation of translation, but completely prevented the formation of polysomes. This implied the crucial importance of the uS19 tail in the elongation process. Analysis of tRNAs associated with 40S subunits and 80S ribosomes containing wild type uS19^FLAG or its truncated form showed that the deletion of the C-terminal pentadecapeptide fragment of uS19 did not interfere with the binding of aminoacyl-tRNA (aa-tRNA) at the ribosomal A site. The results led to the conclusion that the transpeptidation, which occurs on the large ribosomal subunit after decoding the A site codon by the incoming aa-tRNA, is the most likely elongation stage, where this uS19 fragment can play a critical role. Our findings suggest that the uS19 tail is a keystone player in the accommodation of aa-tRNA at the A site, which is a pre-requisite for the peptide transfer.     

The distance between the anticodon loops of two tRNAs bound to the 70 S Escherichia coli ribosome.

Using singlet-singlet energy transfer, we have measured the distance between the anticodons of two transfer RNAs simultaneously bound to a messengerprogramed Escherichia coli 70 S ribosome. The fluorescent Y base adjacent to the anticodon of yeast tRNA_Y^Phe serves as a donor. A proflavine (Pf) chemically substituted for the Y base in tRNA_Pf^Phe serves as an acceptor. By exploiting the sequential binding properties of 70 S ribosomes for two deacylated tRNAs, we can fill the strong site with either tRNA_Y^Phe or tRNA_Pf^Phe and then the weak site with the other tRNA. In both cases donor quenching and sensitized emission of the acceptor are observed. Analysis of these results leads to an estimate for the Y-proflavine distance of 18 ± 2 Å. This distance is very short and suggests strongly that the two tRNAs are simultaneously in contact with adjacent codons of the message. Separate experiments show that binding of a tRNA to the weak site does not perturb the environment of the hypermodified base of a tRNA bound to the strong site. This supports the assignment of the strong site as the peptidyl site. It also indicates that binding of the second tRNA proceeds without a change in the anticodon structure of a pre-existing tRNA at the peptidyl site.     

Identification of a ribosomal ATPase in Escherichia coli cells

Eukaryotic ribosomes harbor an ATPase activity that has been shown to be essential for translation elongation in some lower fungi. Here we report the first identification of a ribosome bound ATPase, RbbA, in E. coli cells. RbbA accounts for most of the ATPase activity associated with 70S ribosomes and 30S ribosomal subunits. Both native and recombinant RbbA were purified and shown to possess ribosome-dependent ATPase activities and to stimulate polyphenylalanine synthesis in vitro. Biochemically, RbbA is similar to the fungi-specific translation elongation factor 3 (EF-3) and cross-reacts with antibody raised against EF-3. The gene encoding RbbA is identified as ORF yhih and the predicted RbbA amino acid sequence is 40% similar to that of the C-terminal half of EF-3. The discovery of a ribosomal ATPase in a prokaryotic cell suggests a common, conserved function for these proteins in translation.     

Translation of poly(U) on ribosomes from Streptomyces aureofaciens

Slowly cooled cells of Streptomyces aureofaciens contained mainly tight-couple ribosomes. Maximum rate of polyphenylalanine synthesis on ribosomes of S. aureofaciens was observed at 40°C, while cultures grew optimally at 28°C. Ribosomes of S. aureofaciens differed from those of E. coli in the amount of poly(U) required for maximum synthetic activity. The polyphenylalanine-synthesizing activity of E. coli ribosomes was about 3-times higher than that of S. aureofaciens ribosomes. The addition of protein S1 of E. coli or the homologous protein from S. aureofaciens had no stimulatory effect on the translation of poly(U). In order to localize alteration(s) of S. aureofaciens ribosomes in the elongation step of polypeptide synthesis we developed an in vitro system derived from purified elongation factors and ribosomal subunits. The enzymatic binding of Phe-tRNA to ribosomes of S. aureofaciens was significantly lower than the binding to ribosomes of E. coli. This alteration was mainly connected with the function of S. aureofaciens 50 S subunits. These subunits were not deficient in their ability to associate with 30 S subunits or with protein SL5 which is homologous to L7/L12 of E. coli.     

Contribution of ribosomal residues to P-site tRNA binding

Structural studies have revealed multiple contacts between the ribosomal P site and tRNA, but how these contacts contribute to P-tRNA binding remains unclear. In this study, the effects of ribosomal mutations on the dissociation rate (k_off) of various tRNAs from the P site were measured. Mutation of the 30S P site destabilized tRNAs to various degrees, depending on the mutation and the species of tRNA. These data support the idea that ribosome-tRNA interactions are idiosyncratically tuned to ensure stable binding of all tRNA species. Unlike deacylated elongator tRNAs, N-acetyl-aminoacyl-tRNAs and tRNA^fMet dissociated from the P site at a similar low rate, even in the presence of various P-site mutations. These data provide evidence for a stability threshold for P-tRNA binding and suggest that ribosome-tRNA^fMet interactions are uniquely tuned for tight binding. The effects of 16S rRNA mutation G1338U were suppressed by 50S E-site mutation C2394A, suggesting that G1338 is particularly important for stabilizing tRNA in the P/E site. Finally, mutation C2394A or the presence of an N-acetyl-aminoacyl group slowed the association rate (k_on) of tRNA dramatically, suggesting that deacylated tRNA binds the P site of the ribosome via the E site.     

Non-enzymic binding of formylmethionyl-transfer RNAf to Artemia salina ribosomes

40 S ribosomal subunits of Artemia salina embryos can bind formylmethionyl-transfer RNA_f non-enzymically, i.e., in the absence of initiation factors. This, like the enzymic reaction, is largely AUG-dependent. Much more fMet-tRNA_f is bound by 80 S ribosomes but, in this case, a large fraction (about two-thirds) of the binding is AUG-independent. Whereas the AUG-dependent binding is very sensitive to edeine, a potent initiation inhibitor, the AUG-independent binding is resistant to this antibiotic. Virtually all of the bound fMet-tRNA_f is in all cases capable of reacting with puromycin to form fMet-puromycin; hence the bound aminoaoyl-tRNA is in the peptidyl (donor) site of the 80 S ribosome. Non-acylated tRNAs also bind to this site with high affinity in a codon-independent reaction and block the 80 S binding of fMet-tRNA_f. The properties of the peptidyl site are consistent with a non-decoding site which harbors the initiator aminoacyl-tRNA, when the 80 S initiation complex is formed, and to which every molecule of tRNA remains temporarily attached following peptide bond synthesis.     

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.     

Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA

In trans-translation transfer messenger RNA (tmRNA) and small protein B (SmpB) rescue ribosomes stalled on truncated or in other ways problematic mRNAs. SmpB promotes the binding of tmRNA to the ribosome but there is uncertainty about the number of participating SmpB molecules as well as their ribosomal location. Here, the interaction of SmpB with ribosomal subunits and ribosomes was studied by isolation of SmpB containing complexes followed by chemical modification of ribosomal RNA with dimethyl sulfate, kethoxal and hydroxyl radicals. The results show that SmpB binds 30S and 50S subunits with 1:1 molar ratios and the 70S ribosome with 2:1 molar ratio. SmpB-footprints are similar on subunits and the ribosome. In the 30S subunit, SmpB footprints nucleotides that are in the vicinity of the P-site facing the E-site, and in the 50S subunit SmpB footprints nucleotides that are located below the L7/L12 stalk in the 3D structure of the ribosome. Based on these results, we suggest a mechanism where two molecules of SmpB interact with tmRNA and the ribosome during trans-translation. The first SmpB molecule binds near the factor-binding site on the 50S subunit helping tmRNA accommodation on the ribosome, whereas the second SmpB molecule may functionally substitute for a missing anticodon stem–loop in tmRNA during later steps of trans-translation.     

GTP-independent tRNA Delivery to the Ribosomal P-site by a Novel Eukaryotic Translation Factor

During translation, aminoacyl-tRNAs are delivered to the ribosome by specialized GTPases called translation factors. Here, we report the tRNA binding to the P-site of 40 S ribosomes by a novel GTP-independent factor eIF2D isolated from mammalian cells. The binding of tRNA_i^Met occurs after the AUG codon finds its position in the P-site of 40 S ribosomes, the situation that takes place during initiation complex formation on the hepatitis C virus internal ribosome entry site or on some other specific RNAs (leaderless mRNA and A-rich mRNAs with relaxed scanning dependence). Its activity in tRNA binding with 40 S subunits does not require the presence of the aminoacyl moiety. Moreover, the factor possesses the unique ability to deliver non-Met (elongator) tRNAs into the P-site of the 40 S subunit. The corresponding gene is found in all eukaryotes and includes an SUI1 domain present also in translation initiation factor eIF1. The versatility of translation initiation strategies in eukaryotes is discussed.     

Structural analysis of 5S rRNA, 5S rRNA-protein complexes and ribosomes employing RNase H and d(GTTCGG)

The hybridization of d(GTTCGG) to eubacterial 5S rRNAs, 5S rRNA-protein complexes, 70S ribosomes and 50S and 30S ribosomal subunits was investigated. This oligonucleotide, which may be considered to be an analogue of the T psi CG loop of tRNAs, was chosen in order to investigate a possible interaction between tRNAs with ribosomal components during protein synthesis. The hybridization was analysed by RNase H hydrolysis studies and, in the case of the ribosomes and ribosomal subunits, in addition with the radioactively labelled oligodeoxyribonucleotide in binding studies. The results obtained lead to the conclusion that nucleotides in loop c, i.e. positions 42-47, are available for oligonucleotide interaction in free Escherichia coli and Bacillus stearothermophilus 5S rRNAs and not available in the corresponding 5S rRNA-protein complexes. The 70S ribosomes and ribosomal subunits did not interact with the oligonucleotide. Under the assumption that d(GTTCGG) is an analogue of the T psi CG loop of tRNAs and in view of the results obtained, we conclude that in the unprogrammed ribosomes the T psi CG loop of tRNAs does not interact via standard Watson-Crick base pairs with the ribosomal 5S, 16S or 23S RNAs.     

The human large subunit ribosomal protein L36A-like contacts the CCA end of P-site bound tRNA

Periodate-oxidized tRNA (tRNAox), the 2′,3′-dialdehyde derivative of tRNA, was used as a zero-length active site-directed affinity labeling reagent, to covalently label proteins at the binding site for the 3′-end of tRNA on human 80S ribosomes. When human 80S ribosomes were reacted with tRNA^Aspox positioned at the P-site, in the presence of an appropriate 12 mer mRNA, a set of two tRNAox-labeled ribosomal proteins (rPs) was observed. The majorily labeled protein was identified as the large subunit rP L36a-like (RPL36AL) by means of mass spectrometry. Intact tRNA^Asp competed with tRNA^Aspox for the binding to the P-site, by preventing tRNA-protein cross-linking with RPL36AL. Altogether, the data presented in this report are consistent with the presence of RPL36AL at or near the binding site for the CCA end of the tRNA substrate positioned at the P-site of human 80S ribosomes. It is the first time that a ribosomal protein is found in an intimate contact (i.e. at a zero-distance) with a nucleotide of the conserved CCA terminus of P-site tRNA which is the substrate of peptidyl transferase reaction. RPL36AL which is strongly conserved in eukaryotes belongs to the L44e family of rPs, a representative of which is Haloarcula marismortui RPL44e.     

Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage

We have recently isolated and characterized a novel protein associated with Escherichia coli ribosomes and named protein Y (pY). Here we show that the ribosomes from bacterial cells growing at a normal physiological temperature contain no pY, whereas a temperature downshift results in the appearance of the protein in ribosomes. The protein also appears in the ribosomes of those cells that reached the stationary phase of growth at a physiological temperature. Our experiments with cell-free translation systems demonstrate that the protein inhibits translation at the elongation stage by blocking the binding of aminoacyl-tRNA to the ribosomal A site. The function of the protein in adaptation of cells to environmental stress is discussed.     

Codon-anticodon interaction at the P site is a prerequisite for tRNA interaction with the small ribosomal subunit

The arrival of high resolution crystal structures for the ribosomal subunits opens a new phase of molecular analysis and asks for corresponding analyses of ribosomal function. Here we apply the phosphorothioate technique to dissect tRNA interactions with the ribosome. We demonstrate that a tRNA bound to the P site of non-programmed 70 S ribosomes contacts predominantly the 50 S, as opposed to the 30 S subunit, indicating that codon-anticodon interaction at the P site is a prerequisite for 30 S binding. Protection patterns of tRNAs bound to isolated subunits and programmed 70 S ribosomes were compared. The results suggest the presence of a movable domain in the large ribosomal subunit that carries tRNA and reveal that only approximately 15% of a tRNA, namely residues 30 +/- 1 to 43 +/- 1, contact the 30 S subunit of programmed 70 S ribosomes, whereas the remaining 85% make contact with the 50 S subunit. Identical protection patterns of two distinct elongator tRNAs at the P site were identified as tRNA species-independent phosphate backbone contacts. The sites of protection correlate nicely with the predicted ribosomal-tRNA contacts deduced from a 5.5-A crystal structure of a programmed 70 S ribosome, thus refining which ribosomal components are critical for tRNA fixation at the P site.     

Identification of the Saccharomyces cerevisiae RNA:pseudouridine synthase responsible for formation of psi(2819) in 21S mitochondrial ribosomal RNA

So far, four RNA:pseudouridine (Ψ)-synthases have been identified in yeast Saccharomyces cerevisiae. Together, they act on cytoplasmic and mitochondrial tRNAs, U2 snRNA and rRNAs from cytoplasmic ribosomes. However, RNA:Ψ-synthases responsible for several U→Ψ conversions in tRNAs and UsnRNAs remained to be identified. Based on conserved amino-acid motifs in already characterised RNA:Ψ-synthases, four additional open reading frames (ORFs) encoding putative RNA:Ψ-synthases were identified in S.cerevisiae. Upon disruption of one of them, the YLR165c ORF, we found that the unique Ψ residue normally present in the fully matured mitochondrial rRNAs (Ψ₂₈₁₉ in 21S rRNA) was missing, while Ψ residues at all the tested pseudouridylation sites in cytoplasmic and mitochondrial tRNAs and in nuclear UsnRNAs were retained. The selective U→Ψ conversion at position 2819 in mitochondrial 21S rRNA was restored when the deleted yeast strain was transformed by a plasmid expressing the wild-type YLR165c ORF. Complementation was lost after point mutation (D71→A) in the postulated active site of the YLR165c-encoded protein, indicating the direct role of the YLR165c protein in Ψ₂₈₁₉ synthesis in mitochondrial 21S rRNA. Hence, for nomenclature homogeneity the YLR165c ORF was renamed PUS5 and the corresponding RNA:Ψ-synthase Pus5p. As already noticed for other mitochondrial RNA modification enzymes, no canonical mitochondrial targeting signal was identified in Pus5p. Our results also show that Ψ₂₈₁₉ in mitochondrial 21S rRNA is not essential for cell viability.