Translational impacts of enzymes that modify ribosomal RNA around the peptidyl transferase centre.

Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the 'critical region' of the peptidyl transferase centre (PTC) in 23S rRNA that exhibited only a minor growth defect at 37°C (although major at 20°C). However, our combined KO of modification enzymes RluC and RlmE (not RluE) resulted in conditional lethality (at 20°C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. In vitro fast kinetics at 20°C and 37°C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Elongation rates of protein synthesis in vivo, as judged by the kinetics of ß-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37°C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg2+. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications per se, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.

Loss of Conserved rRNA Modifications in the Peptidyl Transferase Center Leads to Diminished Protein Synthesis and Cell Growth in Budding Yeast.

Ribosomal RNAs (rRNAs) are extensively modified during the transcription and subsequent maturation. Three types of modifications, 2'-O-methylation of ribose moiety, pseudouridylation, and base modifications, are introduced either by a snoRNA-driven mechanism or by stand-alone enzymes. Modified nucleotides are clustered at the functionally important sites, including peptidyl transferase center (PTC). Therefore, it has been hypothesised that the modified nucleotides play an important role in ensuring the functionality of the ribosome. In this study, we demonstrate that seven 25S rRNA modifications, including four evolutionarily conserved modifications, in the proximity of PTC can be simultaneously depleted without loss of cell viability. Yeast mutants lacking three snoRNA genes (snR34, snR52, and snR65) and/or expressing enzymatically inactive variants of spb1(D52A/E679K) and nop2(C424A/C478A) were constructed. The results show that rRNA modifications in PTC contribute collectively to efficient translation in eukaryotic cells. The deficiency of seven modified nucleotides in 25S rRNA resulted in reduced cell growth, cold sensitivity, decreased translation levels, and hyperaccurate translation, as indicated by the reduced missense and nonsense suppression. The modification m5C2870 is crucial in the absence of the other six modified nucleotides. Thus, the pattern of rRNA-modified nucleotides around the PTC is essential for optimal ribosomal translational activity and translational fidelity.

SILAC analysis of Escherichia coli proteome during progression of growth from exponential to prolonged stationary phase.

The expression level of individual proteins varies markedly during the progression of the growth phase in bacteria. A set of proteins was quantified in Escherichia coli total proteome during 14 days of batch cultivation using pulse stable isotope labeled amino acids in cell culture (SILAC)-based quantitative mass spectrometry.

Ribosomal RNA modification enzymes stimulate large ribosome subunit assembly in E. coli.

Ribosomal RNA modifications are introduced by specific enzymes during ribosome assembly in bacteria. Deletion of individual modification enzymes has a minor effect on bacterial growth, ribosome biogenesis, and translation, which has complicated the definition of the function of the enzymes and their products. We have constructed an Escherichia coli strain lacking 10 genes encoding enzymes that modify 23S rRNA around the peptidyl-transferase center. This strain exhibits severely compromised growth and ribosome assembly, especially at lower temperatures. Re-introduction of the individual modification enzymes allows for the definition of their functions. The results demonstrate that in addition to previously known RlmE, also RlmB, RlmKL, RlmN and RluC facilitate large ribosome subunit assembly. RlmB and RlmKL have functions in ribosome assembly independent of their modification activities. While the assembly stage specificity of rRNA modification enzymes is well established, this study demonstrates that there is a mutual interdependence between the rRNA modification process and large ribosome subunit assembly.

Ribosome Protein Composition Mediates Translation during the Escherichia coli Stationary Phase.

Bacterial ribosomes contain over 50 ribosome core proteins (r-proteins). Tens of non-ribosomal proteins bind to ribosomes to promote various steps of translation or suppress protein synthesis during ribosome hibernation. This study sets out to determine how translation activity is regulated during the prolonged stationary phase. Here, we report the protein composition of ribosomes during the stationary phase. According to quantitative mass-spectrometry analysis, ribosome core proteins bL31B and bL36B are present during the late log and first days of the stationary phase and are replaced by corresponding A paralogs later in the prolonged stationary phase. Ribosome hibernation factors Rmf, Hpf, RaiA, and Sra are bound to the ribosomes during the onset and a few first days of the stationary phase when translation is strongly suppressed. In the prolonged stationary phase, a decrease in ribosome concentration is accompanied by an increase in translation and association of translation factors with simultaneous dissociation of ribosome hibernating factors. The dynamics of ribosome-associated proteins partially explain the changes in translation activity during the stationary phase.

Variance in translational fidelity of different bacterial species is affected by pseudouridines in the tRNA anticodon stem-loop.

Delicate variances in the translational machinery affect how efficiently different organisms approach protein synthesis. Determining the scale of this effect, however, requires knowledge on the differences of mistranslation levels. Here, we used a dual-luciferase reporter assay cloned into a broad host range plasmid to reveal the translational fidelity profiles of Pseudomonas putida, Pseudomonas aeruginosa and Escherichia coli. We observed that these profiles are surprisingly different, whereas species more prone to translational frameshifting are not necessarily more prone to stop codon readthrough. As tRNA modifications are among the factors that have been implicated to affect translation accuracy, we also show that translational fidelity is context-specifically influenced by pseudouridines in the anticodon stem-loop of tRNA, but the effect is not uniform between species.

A Conundrum of r-Protein Stability: Unbalanced Stoichiometry of r-Proteins during Stationary Phase in Escherichia coli.

Bacterial ribosomes are composed of three rRNA and over 50 ribosomal protein (r-protein) molecules. r-proteins are essential for ribosome assembly and structural stability and also participate in almost all ribosome functions. Ribosomal components are present in stoichiometric amounts in the mature 70S ribosomes during exponential and early stationary growth phases. Ribosomes are degraded in stationary phase; however, the stability and fate of r-proteins during stationary growth phase are not known. In this study, we report a quantitative analysis of ribosomal components during extended stationary-phase growth in Escherichia coli. We show that (i) the quantity of ribosomes per cell mass decreases in stationary phase, (ii) 70S ribosomes contain r-proteins in stoichiometric amounts, (iii) 30S subunits are degraded faster than 50S subunits, (iv) the quantities of 21 r-proteins in the total proteome decrease during 14 days (short-lived r-proteins) concomitantly with the reduction of cellular RNA, and (e) 30 r-proteins are stable and form a pool of free r-proteins (stable r-proteins). Thus, r-proteins are present in nonstoichiometric amounts in the proteome of E. coli during the extended stationary phase. IMPORTANCE Ribosome degradation has been extensively described from the viewpoint of its main component, rRNA. Here, we aim to complement our knowledge by quantitatively analyzing r-protein degradation and stability both in the ribosomes and in the whole-cell proteome during stationary phase in E. coli. r-proteins are considered to be very stable in the proteome. Here, we show that a specific set of r-proteins are rapidly degraded after release from the rRNA. The degradation of r-proteins is an intriguing new aspect of r-protein metabolism in bacteria.

Plasticity and conditional essentiality of modification enzymes for domain V of Escherichia coli 23S ribosomal RNA.

Escherichia coli rRNAs are post-transcriptionally modified at 36 positions but their modification enzymes are dispensable individually for growth, bringing into question their significance. However, a major growth defect was reported for deletion of the RlmE enzyme, which abolished a 2'O methylation near the peptidyl transferase center (PTC) of the 23S rRNA. Additionally, an adjacent 80-nt "critical region" around the PTC had to be modified to yield significant peptidyl transferase activity in vitro. Surprisingly, we discovered that an absence of just two rRNA modification enzymes is conditionally lethal (at 20°C): RlmE and RluC. At a permissive temperature (37°C), this double knockout was shown to abolish four modifications and be defective in ribosome assembly, though not more so than the RlmE single knockout. However, the double knockout exhibited an even lower rate of tripeptide synthesis than did the single knockout, suggesting an even more defective ribosomal translocation. A combination knockout of the five critical-region-modifying enzymes RluC, RlmKL, RlmN, RlmM, and RluE (not RlmE), which synthesize five of the seven critical-region modifications and 14 rRNA and tRNA modifications altogether, was viable (minor growth defect at 37°C, major at 20°C). This was surprising based on prior in vitro studies. This five-knockout combination had minimal effects on ribosome assembly and frameshifting at 37°C, but greater effects on ribosome assembly and in vitro peptidyl transferase activity at cooler temperatures. These results establish the conditional essentiality of bacterial rRNA modification enzymes and also reveal unexpected plasticity of modification of the PTC region in vivo.

Luciferase-based reporter system for in vitro evaluation of elongation rate and processivity of ribosomes.

The elongation step of translation is a key contributor to the abundance, folding and quality of proteins and to the stability of mRNA. However, control over translation elongation has not been thoroughly investigated. In this study, a Renilla-firefly luciferase fusion reporter system was further developed to investigate the in vitro elongation rate and processivity of ribosomes independent of the initiation and termination steps. The reporter mRNA was constructed to contain a single ORF encoding in-frame Renilla luciferase, a specific domain moiety and firefly luciferase. Such a reporter structure enables the quantitative and individual evaluation of the synthesis of a specific domain. As a proof of principle, the synthesis of three protein domains of different lengths and structures was analyzed. Using a cell-free translation assay, both the elongation rate and processivity of ribosomes were shown to vary depending on the domain synthesized. Additionally, a stalling sequence consisting of ten rare arginine codons notably reduced the elongation rate and the processivity of the ribosomes. All these results are consistent with the previously known dynamics of elongation in vivo. Overall, the methodology presented in this report provides a framework for studying aspects that contribute to the elongation step of translation.

A mutation in the ribosomal protein uS12 reveals novel functions of its universally conserved PNSA loop.

The ribosomal protein uS12 is conserved across all domains of life. Recently, a heterozygous spontaneous mutation in human uS12 (corresponding to R49K mutation immediately downstream of the universally conserved 44 PNSA47 loop in Escherichia coli uS12) was identified for causing ribosomopathy, highlighting the importance of the PNSA loop. To investigate the effects of a similar mutation in the absence of any wild-type alleles, we mutated the rpsL gene (encoding uS12) in E. coli. Consistent with its pathology (in humans), we were unable to generate the R49K mutation in E. coli in the absence of a support plasmid. However, we were able to generate the L48K mutation in its immediate vicinity. The L48K mutation resulted in a cold sensitive phenotype and ribosome biogenesis defect in the strain. We show that the L48K mutation impacts the steps of initiation and elongation. Furthermore, the genetic interactions of the L48K mutation with RRF and Pth suggest a novel role of the PNSA loop in ribosome recycling. Our studies reveal new functions of the PNSA loop in uS12, which has so far been studied in the context of translation elongation.

Pseudouridines of tRNA Anticodon Stem-Loop Have Unexpected Role in Mutagenesis in Pseudomonas sp.

Pseudouridines are known to be important for optimal translation. In this study we demonstrate an unexpected link between pseudouridylation of tRNA and mutation frequency in Pseudomonas species. We observed that the lack of pseudouridylation activity of pseudouridine synthases TruA or RluA elevates the mutation frequency in Pseudomonas putida 3 to 5-fold. The absence of TruA but not RluA elevates mutation frequency also in Pseudomonas aeruginosa. Based on the results of genetic studies and analysis of proteome data, the mutagenic effect of the pseudouridylation deficiency cannot be ascribed to the involvement of error-prone DNA polymerases or malfunctioning of DNA repair pathways. In addition, although the deficiency in TruA-dependent pseudouridylation made P. putida cells more sensitive to antimicrobial compounds that may cause oxidative stress and DNA damage, cultivation of bacteria in the presence of reactive oxygen species (ROS)-scavenging compounds did not eliminate the mutator phenotype. Thus, the elevated mutation frequency in the absence of tRNA pseudouridylation could be the result of a more specific response or, alternatively, of a cumulative effect of several small effects disturbing distinct cellular functions, which remain undetected when studied independently. This work suggests that pseudouridines link the translation machinery to mutation frequency.

Phenotypic effects of paralogous ribosomal proteins bL31A and bL31B in E. coli.

Ribosomes are essential macromolecular complexes conducting protein biosynthesis in all domains of life. Cells can have heterogeneous ribosomes, i.e. ribosomes with various ribosomal RNA and ribosomal protein (r-protein) composition. However, the functional importance of heterogeneous ribosomes has remained elusive. One of the possible sources for ribosome heterogeneity is provided by paralogous r-proteins. In E. coli, ribosomal protein bL31 has two paralogs: bL31A encoded by rpmE and bL31B encoded by ykgM. This study investigates phenotypic effects of these ribosomal protein paralogs using bacterial strains expressing only bL31A or bL31B. We show that bL31A confers higher fitness to E. coli under lower temperatures. In addition, bL31A and bL31B have different effects on translation reading frame maintenance and apparent translation processivity in vivo as demonstrated by dual luciferase assay. In general, this study demonstrates that ribosomal protein paralog composition (bL31A versus bL31B) can affect cell growth and translation outcome.

Ribosome engineering reveals the importance of 5S rRNA autonomy for ribosome assembly.

5S rRNA is an indispensable component of cytoplasmic ribosomes in all species. The functions of 5S rRNA and the reasons for its evolutionary preservation as an independent molecule remain unclear. Here we used ribosome engineering to investigate whether 5S rRNA autonomy is critical for ribosome function and cell survival. By linking circularly permutated 5S rRNA with 23S rRNA we generated a bacterial strain devoid of free 5S rRNA. Viability of the engineered cells demonstrates that autonomous 5S rRNA is dispensable for cell growth under standard conditions and is unlikely to have essential functions outside the ribosome. The fully assembled ribosomes carrying 23S-5S rRNA are highly active in translation. However, the engineered cells accumulate aberrant 50S subunits unable to form stable 70S ribosomes. Cryo-EM analysis revealed a malformed peptidyl transferase center in the misassembled 50S subunits. Our results argue that the autonomy of 5S rRNA is preserved due to its role in ribosome biogenesis.

Functional Interactions of Ribosomal Intersubunit Bridges in Saccharomyces cerevisiae.

Ribosomes of Archaea and Eukarya share higher homology with each other than with bacterial ribosomes. For example, there is a set of 35 r-proteins that are specific only for archaeal and eukaryotic ribosomes. Three of these proteins-eL19, eL24, and eL41-participate in interactions between ribosomal subunits. The eukaryote-specific extensions of r-proteins eL19 and eL24 form two intersubunit bridges eB12 and eB13, which are present only in eukaryotic ribosomes. The third r-protein, eL41, forms bridge eB14. Notably, eL41 is found in all eukaryotes but only in some Archaea. It has been shown that bridges eB12 and eB13 are needed for efficient translation, while r-protein eL41 plays a minor role in ribosome function. Here, the functional interactions between intersubunit bridges were studied using budding yeast strains lacking different combinations of the abovementioned bridges/proteins. The growth phenotypes, levels of in vivo translation, ribosome-polysome profiles, and in vitro association of ribosomal subunits were analyzed. The results show a genetic interaction between r-protein eL41 and the eB12 bridge-forming region of eL19, and between r-proteins eL41 and eL24. It was possible to construct viable yeast strains with Archaea-like ribosomes lacking two or three eukaryote-specific bridges. These strains display slow growth and a poor translation phenotype. In addition, bridges eB12 and eB13 appear to cooperate during ribosome subunit association. These results indicate that nonessential structural elements of r-proteins become highly important in the context of disturbed subunit interactions. Therefore, eukaryote-specific bridges may contribute to the evolutionary success of eukaryotic translation machinery.

Assembly and functionality of the ribosome with tethered subunits.

Ribo-T is an engineered ribosome whose small and large subunits are tethered together by linking 16S rRNA and 23S rRNA in a single molecule. Although Ribo-T can support cell proliferation in the absence of wild type ribosomes, Ribo-T cells grow slower than those with wild type ribosomes. Here, we show that cell growth defect is likely explained primarily by slow Ribo-T assembly rather than its imperfect functionality. Ribo-T maturation is stalled at a late assembly stage. Several post-transcriptional rRNA modifications and some ribosomal proteins are underrepresented in the accumulated assembly intermediates and rRNA ends are incompletely trimmed. Ribosome profiling of Ribo-T cells shows no defects in translation elongation but reveals somewhat higher occupancy by Ribo-T of the start codons and to a lesser extent stop codons, suggesting that subunit tethering mildly affects the initiation and termination stages of translation. Understanding limitations of Ribo-T system offers ways for its future development.

Pseudomonas putida Responds to the Toxin GraT by Inducing Ribosome Biogenesis Factors and Repressing TCA Cycle Enzymes.

The potentially self-poisonous toxin-antitoxin modules are widespread in bacterial chromosomes, but despite extensive studies, their biological importance remains poorly understood. Here, we used whole-cell proteomics to study the cellular effects of the Pseudomonas putida toxin GraT that is known to inhibit growth and ribosome maturation in a cold-dependent manner when the graA antitoxin gene is deleted from the genome. Proteomic analysis of P. putida wild-type and ΔgraA strains at 30 °C and 25 °C, where the growth is differently affected by GraT, revealed two major responses to GraT at both temperatures. First, ribosome biogenesis factors, including the RNA helicase DeaD and RNase III, are upregulated in ΔgraA. This likely serves to alleviate the ribosome biogenesis defect of the ΔgraA strain. Secondly, proteome data indicated that GraT induces downregulation of central carbon metabolism, as suggested by the decreased levels of TCA cycle enzymes isocitrate dehydrogenase Idh, α-ketoglutarate dehydrogenase subunit SucA, and succinate-CoA ligase subunit SucD. Metabolomic analysis revealed remarkable GraT-dependent accumulation of oxaloacetate at 25 °C and a reduced amount of malate, another TCA intermediate. The accumulation of oxaloacetate is likely due to decreased flux through the TCA cycle but also indicates inhibition of anabolic pathways in GraT-affected bacteria. Thus, proteomic and metabolomic analysis of the ΔgraA strain revealed that GraT-mediated stress triggers several responses that reprogram the cell physiology to alleviate the GraT-caused damage.

Ribosomal protein eL24, involved in two intersubunit bridges, stimulates translation initiation and elongation.

Interactions between subunits in the Saccharomyces cerevisiae ribosome are mediated by universal and eukaryote-specific intersubunit bridges. Universal bridges are positioned close to the ribosomal functional centers, while eukaryote-specific bridges are mainly located on the periphery of the ribosome. Two bridges, eB13 and B6, are formed by the ribosomal protein eL24. The eukaryotic eL24 is composed of an N-terminal domain, a linker region and a C-terminal α-helix. Here, the functions of different domains of eL24 in the S. cerevisiae ribosome were evaluated. The C-terminal domain and the linker region of the eL24 form eukaryote-specific eB13 bridge. Phenotypic characterization of the eL24 deletion mutants indicated that the functional integrity of the eB13 bridge mainly depends on the protein-protein contacts between eL24 and eS6. Further investigation showed importance of the eB13 bridge in the subunit joining in vivo and in vitro. In vitro translation assay demonstrated the role of the eB13 bridge in both initiation and elongation steps of translation. Intriguingly, results of in vitro translation experiment suggest involvement of the N-terminal domain of eL24 in the translation initiation. Therefore, eL24 performs number of tasks required for the optimal ribosome functionality.

Bacterial ribosome heterogeneity: Changes in ribosomal protein composition during transition into stationary growth phase.

Ribosomes consist of many small proteins and few large RNA molecules. Both components are necessary for ribosome functioning during translation. According to widely accepted view, bacterial ribosomes contain always the same complement of ribosomal proteins. Comparative bacterial genomics data indicates that several ribosomal proteins are encoded by multiple paralogous genes suggesting structural heterogeneity of ribosomes. In E. coli, two r-proteins bL31 and bL36 are encoded by two genes: rpmE and ykgM encode bL31 protein paralogs bL31A and bL31B, and rpmJ and ykgO encode bL36 protein paralogs bL36A and bL36B respectively. We have found several similarities and differences between ribosomes of exponential and stationary growth phases by using quantitative mass spectrometry and X-ray crystallography. First, composition of ribosome associating proteins changes profoundly as cells transition from exponential to stationary growth phase. Ribosomal core proteins bL31A and bL36A are replaced by bL31B and bL36B, respectively. Second, our X-ray structure of the 70S ribosome demonstrates that bL31B and bL36B proteins have similar ribosome binding sites to their A counterparts. Third, ribosome subpopulations containing A or B paralogs existed simultaneously demonstrating that E. coli ribosomes are heterogeneous with respect to their paralogous ribosomal protein composition that changes via protein exchange.

Pseudouridine-Free Escherichia coli Ribosomes.

Pseudouridine (Ψ) is present at conserved, functionally important regions in the ribosomal RNAs (rRNAs) from all three domains of life. Little, however, is known about the functions of Ψ modifications in bacterial ribosomes. An Escherichia coli strain has been constructed in which all seven rRNA Ψ synthases have been inactivated and whose ribosomes are devoid of all Ψs. Surprisingly, this strain displays only minor defects in ribosome biogenesis and function, and cell growth is only modestly affected. This is in contrast to a strong requirement for Ψ in eukaryotic ribosomes and suggests divergent roles for rRNA Ψ modifications in these two domains.IMPORTANCE Pseudouridine (Ψ) is the most abundant posttranscriptional modification in RNAs. In the ribosome, Ψ modifications are typically located at conserved, critical regions, suggesting they play an important functional role. In eukarya and archaea, rRNAs are modified by a single pseudouridine synthase (PUS) enzyme, targeted to rRNA via a snoRNA-dependent mechanism, while bacteria use multiple stand-alone PUS enzymes. Disruption of Ψ modification of rRNA in eukarya seriously impairs ribosome function and cell growth. We have constructed an E. coli multiple deletion strain lacking all Ψ modifications in rRNA. In contrast to the equivalent eukaryotic mutants, the E. coli strain is only modestly affected in growth, decoding, and ribosome biogenesis, indicating a differential requirement for Ψ modifications in these two domains.

Random pseuoduridylation in vivo reveals critical region of Escherichia coli 23S rRNA for ribosome assembly.

Pseudouridine is the most common modified nucleoside in RNA, which is found in stable RNA species and in eukaryotic mRNAs. Functional analysis of pseudouridine is complicated by marginal effect of its absence. We demonstrate that excessive pseudouridines in rRNA inhibit ribosome assembly. Ten-fold increase of pseudouridines in the 16S and 23S rRNA made by a chimeric pseudouridine synthase leads to accumulation of the incompletely assembled large ribosome subunits. Hyper modified 23S rRNA is found in the r-protein assembly defective particles and are selected against in the 70S and polysome fractions showing modification interference. Eighteen positions of 23S rRNA were identified where isomerization of uridines interferes with ribosome assembly. Most of the interference sites are located in the conserved core of the large subunit, in the domain 0 of 23S rRNA, around the peptide exit tunnel. A plausible reason for pseudouridine-dependent inhibition of ribosome assembly is stabilization of rRNA structure, which leads to the folding traps of rRNA and to the retardation of the ribosome assembly.