TABLE 2 - uploaded by Jaanus Remme
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Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA dur...
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... A 260 /A 280 ratio was used to confirm the peak identities. The RP- HPLC peak surface area corresponding to each nucleoside was calculated and compared with the respective nucleoside peak area of mature 16S rRNA of 70S ribosomes ( Fig. 3; Table 2). Mature 16S rRNA isolated from 70S ribosomes has been shown to contain stochiometric amounts of modified nucleosides within 10% error limit (Gehrke and Kuo 1989). ...
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... 25S particles formed in the pres- ence of Cam or Ery contain only 16S rRNA that is incompletely processed (Siibak et al. 2009). 16S rRNA of both Cam and Ery 25S particles clearly contain several modified nucleosides, albeit at a lower level compared with the mature 30S particles ( Fig. 3; Table 2). Both Cam and Ery 25S particles have similar nucle- oside composition ( Fig. 3; Table 2). ...
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... rRNA of both Cam and Ery 25S particles clearly contain several modified nucleosides, albeit at a lower level compared with the mature 30S particles ( Fig. 3; Table 2). Both Cam and Ery 25S particles have similar nucle- oside composition ( Fig. 3; Table 2). Pseudouridine, m 5 C, and m 4 Cm are present in 25S particles between 40% and 60% compared with the mature 16S rRNA. ...
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... 7 G and m 2 G are found in 25S particles in <25%. m 5 U and m 6 2 A are found in 25S particles only in trace amounts ( Table 2). The results were well reproducible, except in m 4 Cm, which exhibited significant variation in different preparations. ...
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... the majority of these modifications are introduced into 16S rRNA during intermediate stages of small subunit assembly. Formation of m 4 Cm seems also to occur during intermediate assembly stages, although only 70% was found in the free 30S fraction and a large variability of results was observed (Table 2). m 5 U and m 6 2 A are present in the free 30S particles at z20% level (Table 2), which is about the same fraction as the mature 59 end of 16S was found. ...
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... of m 4 Cm seems also to occur during intermediate assembly stages, although only 70% was found in the free 30S fraction and a large variability of results was observed (Table 2). m 5 U and m 6 2 A are present in the free 30S particles at z20% level (Table 2), which is about the same fraction as the mature 59 end of 16S was found. Therefore, m 5 U and m 6 2 A can be classified as late assembly specific modifications. ...
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... residues were quantitated by RP HPLC. According to the HPLC analysis, 35S particles contain 65%-70%, 45S particles contain 80%, and free 50S subunits contain 90% of pseu- douridines compared with the 23S rRNA of 70S ribosomes (Table 2). It must be taken into account that the methylated pseudouridine (m 3 C) co-elutes with m 5 C and therefore does not contribute to the pseudouridine peak. ...
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... m 5 C residue at position 1962 of E. coli 23S rRNA is the product of RlmI (YccW) ( Purta et al. 2008). The peak at 11.4 min of the HPLC derived from 35S, 45S particles, and from free 50S subunits constitutes 40%, 50%, and 70%, respectively ( Fig. 3; Table 2). This peak contains two nucleosides m 5 C and m 3 C ( Gehrke and Kuo 1989). ...
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Citations
... Since these intermediates represent distinct stages and pathways of large subunit ribosome assembly, our experiments identify the specific stages in these pathways at which m 1 G, m 2 G, m 7 G, and D modifications are incorporated into the 23S rRNA. Furthermore, the stages of large subunit ribosome assembly during which the D modification is incorporated into E. coli 23S rRNA have not been determined under any cellular or environmental conditions [52][53][54] . Therefore, this is the first study to determine the specific stages during large subunit ribosome assembly at which the D modification is incorporated into E. coli 23S rRNA. ...
... . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Previous studies have found that in E. coli cells exposed to erythromycin and chloramphenicol, in cells lacking the DEAD-box RNA helicase SrmB, and in wild-type cells, the RlmA and RlmG methyltransferases function during the early stages of large subunit ribosome assembly [52][53][54] . ...
... Furthermore, in cells exposed to erythromycin or chloramphenicol, the RlmL enzyme was found to act during the early stages of large subunit ribosome assembly 52 . However, no distinctions were made in the above studies between very-early and early stages of large-subunit ribosome assembly [52][53][54] . Thus, in our cells expressing the helicase-inactive R331A DbpA construct, in the pathways where the 27S and 35S intermediates accumulate, the RlmA, RlmG, and RlmL enzymes act during the early stages of ribosome assembly, similar to previously investigated cellular or stress conditions [52][53][54] . ...
RNA post-transcriptional modifications are ubiquitous across all organisms and serve as fundamental regulators of cellular homeostasis, growth, and stress adaptation. Techniques for the simultaneous detection of multiple RNA modifications in a high-throughput, single-nucleotide-resolution manner are largely absent in the field, and developing such techniques is of paramount importance. We used the Escherichia coli ribosome as a model system to develop novel techniques for RNA post-transcriptional modification detection, leveraging its extensive and diverse array of modifications. For modification detection, we performed reverse transcriptase reactions in the presence of Mn ²⁺ and quantified the reverse transcriptase deletions and misincorporations at modification positions using Illumina next-generation sequencing. We simultaneously detected the following modifications in ribosomal RNA (rRNA): 1-methylguanosine (m ¹ G), 2-methylguanosine (m ² G), 3-methylpseudouridine, N ⁶ ,N ⁶ -dimethyladenosine, and 3-methyluridine, without chemical treatment. Furthermore, subjecting the rRNA samples to 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho- p -toluenesulfonate followed by alkaline conditions allowed us to simultaneously detect pseudouridine, 7-methylguanosine (m ⁷ G), 5-hydroxycytidine (OH ⁵ C), 2-methyladenosine, and dihydrouridine (D). Finally, subjecting the rRNA samples to KMnO 4 followed by alkaline conditions allowed us to simultaneously detect m ⁷ G, OH ⁵ C, and D. Our results reveal that m ¹ G, m ² G, m ⁷ G, and D are incorporated prior to the accumulation of the 27S, 35S, and 45S large subunit intermediates in cells expressing the helicase-inactive R331A DbpA construct. These intermediates belong to three distinct stages and pathways of large subunit ribosome assembly. Therefore, our results identify the time points in three pathways at which m ¹ G, m ² G, m ⁷ G, and D are incorporated into the large ribosome subunit and provide a framework for broader studies on RNA modification dynamics.
... CAM has the ability to bind to the PTC and consequently affect translation [25]. To systematically investigate the impact of CAM on the translation process within cells, we first conducted an LFQ-MS analysis of protein levels in E. coli MG1655 strains with 7 µg/mL pf CAM for 2h at 25 • C (termed CAM + ), as previously described [35]. In E. coli, ribosome biogenesis tends to be more sensitive at lower temperatures, and the choice of 25 • C helps in capturing potential defects in ribosome assembly that might not be evident at higher temperatures [36]. ...
... To investigate the impact of CAM on this process, we employed a sucrose gradient to isolate the ribosomal precursors from the E. coli MG1655 strain, which was treated with CAM under the same conditions as in the LFQ-MS experiment (Figure 2A,B). Consistent with previous studies [35], precursors of the ribosomal small subunit (25S) as well as those of the large subunit (35S and 45S) were identified ( Figure 2B). Peaks were further inspected by cryo-EM or negative staining, which showed good quality for 35S and 45S peaks ( Figure S3A,B). ...
Chloramphenicol (CAM), a well-known broad-spectrum antibiotic, inhibits peptide bond formation in bacterial ribosomes. It has been reported to affect ribosome assembly mainly through disrupting the balance of ribosomal proteins. The present study investigates the multifaceted effects of CAM on the maturation of the 50S ribosomal subunit in Escherichia coli (E. coli). Using label-free quantitative mass spectrometry (LFQ-MS), we observed that CAM treatment also leads to the upregulation of assembly factors. Further cryo-electron microscopy (cryo-EM) analysis of the ribosomal precursors characterized the CAM-treatment-accumulated pre-50S intermediates. Heterogeneous reconstruction identified 26 distinct pre-50S intermediates, which were categorized into nine main states based on their structural features. Our structural analysis highlighted that CAM severely impedes the formation of the central protuberance (CP), H89, and H58 during 50S ribosomal subunit maturation. The ELISA assay further demonstrated the direct binding of CAM to the ribosomal precursors, suggesting that the interference with 50S maturation occurs through a combination of direct and indirect mechanisms. These findings provide new insights into the mechanism of the action of CAM and provide a foundation for a better understanding of the assembly landscapes of the ribosome.
... Over the years, extensive studies have identified various post-transcriptional modifications that adorn rRNA molecules. These modifications, which are added during ribosome assembly (Siibak and Remme 2010), participate in the balance between translation speed and accuracy. Indeed, in vitro reconstituted Escherichia coli ribosomes methyltransferase, RlmP, methylates G2553 during 50S biogenesis (Hansen et al. 2002;Roovers et al. 2022). ...
rRNA modifications play crucial roles in fine-tuning the delicate balance between translation speed and accuracy, yet the underlying mechanisms remain elusive. Comparative analyses of the ribosomal RNA modifications in taxonomically distant bacteria could help define their general, as well as species-specific, roles. In this study, we identified a new methyltransferase, RlmQ, in Staphylococcus aureus responsible for the Gram-positive specific m ⁷ G2601, which is not modified in E. coli (G2574). We also demonstrate the absence of methylation on C1989, equivalent to E. coli C1962, which is methylated at position 5 by the Gram-negative specific RlmI methyltransferase, a paralogue of RlmQ. Both modifications ( S. aureus m ⁷ G2601 and E. coli m ⁵ C1962) are situated within the same tRNA accommodation corridor, hinting at a potential shared function in translation. Inactivation of S. aureus rlm Q causes the loss of methylation at G2601 and significantly impacts growth, cytotoxicity, and biofilm formation. These findings unravel the intricate connections between rRNA modifications, translation, and virulence in pathogenic Gram-positive bacteria.
... Some enzymes need ribosomal proteins to carry out modifications while other enzymes only modify while rRNA is protein free. In vitro, sub-ribosomal particle analysis has shown that most methylations occur during the early or late steps of ribosome assembly [4]. Various Pseudouridine synthases carry out pseudouridylation. ...
Background
Ribosomal RNA modifications are pivotal in bacteria. A method for locating rRNA modification sites and associated enzymes is crucial for understanding ribosome formation, bacterial functionality, and antibiotic resistance correlated with rRNA modifications. ModrRNA1 introduces a bioinformatics approach to streamline this identification process by replacing manual methods and combining all the required computational steps in a user-friendly web tool. It may aid in locating potential uncharacterized modification sites and previously unknown RNA modification enzymes across various bacteria, enhancing the comprehension of their biological importance.
Availability and implementation
ModrRNA1 employs a curated database of known bacterial rRNA modification sequences and associated enzymes. Sequence alignment, with a customized scoring system, enables accurate general identification of modification sites. High-confidence alignments generate annotated plots over the RNA sequence. Subsequent enzyme detection involves structural comparisons, phylogenetic assessments, and BLAST searches, aiding in enzyme identification. The tool boasts a user-friendly web interface and is hosted on Azure, offering accessible rRNA modification analysis. It is available as a web tool on https://modrrna.biotechie.org and the source code is freely available at https://github.com/sciguysl/ModrRNA1 for any customization purposes.
Conclusion
The results demonstrate the effectiveness of ModrRNA1 in identifying potential non-species-specific rRNA modification sites in different bacteria and their associated enzymes specific to the query bacteria. It can be utilized to identify uncharacterized rRNA modification sites and undiscovered proteins with RNA modifying activity.
... Over the years, extensive studies have identified various post-transcriptional modifications that adorn rRNA molecules. These modifications, which are added during ribosome assembly (Siibak and Remme 2010), participate in the balance between translation speed and accuracy. Indeed, in vitro reconstituted Escherichia coli ribosomes (which was not certified by peer review) is the author/funder. ...
rRNA modifications play crucial roles in fine-tuning the delicate balance between translation speed and accuracy, yet the underlying mechanisms remain elusive. Comparative analysis of the ribosomal RNA modifications in taxonomically distant bacteria could help define their general as well as species-specific roles. In this study, we identified a new methyltransferase, RlmQ, in Staphylococcus aureus responsible for the Gram-positive specific m ⁷ G2601, which is not modified in E. coli (G2574). We also demonstrate the absence of methylation on C1989, equivalent to E. coli C1962, which is methylated at position 5 by the Gram-negative specific RlmI methyltransferase, a paralogue of RlmQ. Both modifications ( S. aureus m ⁷ G2601 and E. coli m ⁵ C1962) are situated within the same tRNA accommodation corridor, hinting at a potential shared function in translation. Inactivation of S. aureus rlm Q causes the loss of methylation at G2601 and significantly impacts growth, cytotoxicity, and biofilm formation. These findings unravel the intricate connections between rRNA modifications, translation, and virulence in pathogenic Gram-positive bacteria.
... These modifications are deposited site-specifically by multiple different modification enzymes during the course of the assembly process. Traditionally, modifications are detected using reverse transcriptase primer extension techniques [61], or P1 nuclease digestion followed by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC) [62,63]. Although these are very sensitive methods, they are tedious as they allow for the observation of only one modification at a time and are suitable for detecting only specific modifications. ...
Ribosome assembly is one of the most fundamental processes of gene expression and has served as a playground for investigating the molecular mechanisms of how protein–RNA complexes (RNPs) assemble. A bacterial ribosome is composed of around 50 ribosomal proteins, several of which are co-transcriptionally assembled on a ~4500-nucleotide-long pre-rRNA transcript that is further processed and modified during transcription, the entire process taking around 2 min in vivo and being assisted by dozens of assembly factors. How this complex molecular process works so efficiently to produce an active ribosome has been investigated over decades, resulting in the development of a plethora of novel approaches that can also be used to study the assembly of other RNPs in prokaryotes and eukaryotes. Here, we review biochemical, structural, and biophysical methods that have been developed and integrated to provide a detailed and quantitative understanding of the complex and intricate molecular process of bacterial ribosome assembly. We also discuss emerging, cutting-edge approaches that could be used in the future to study how transcription, rRNA processing, cellular factors, and the native cellular environment shape ribosome assembly and RNP assembly at large.
... These modifications are deposited site-specifically by multiple different modification enzymes during the course of the assembly process. Traditionally modifications are detected using reverse transcriptase primer extension techniques [61], P1 nuclease digestion followed by Thin-Layer Chromatography (TLC) or High Performance Liquid Chromatography (HPLC) [62,63]. Although these are very sensitive methods, they are tedious as they allow the observation of only one modification at a time and are suitable to detect only specific modifications. ...
Ribosome assembly is one of the most fundamental processes in gene expression and has served as a playground to investigate the molecular mechanisms of how protein-RNA complexes (RNPs) assemble. The bacterial ribosome is composed of around 50 ribosomal proteins several of which are co-transcriptionally assembled on a ~4,500 nucleotides long pre-rRNA transcript that is further processed and modified during transcription, the entire process taking around 2 minutes in vivo and assisted by dozens of assembly factors. How this complex molecular process works so efficiently to produce an active ribosome has been investigated over decades and has resulted in the development of a plethora of novel approaches that can also be used to study the assembly of other RNPs. Here we review biochemical, structural and biophysical methods that have been developed and integrated to provide a detailed and quantitative understanding of this complex and intricate molecular process of assembly. We also discuss emerging cutting-edge approaches that could be used in the future to study how transcription, rRNA processing, cellular factors and the native cellular environment shape ribosome assembly and RNP assembly at large.
... The reconstructed series of particles subjected to r-protein dissociation corroborates the multi-pathway of assembly [13,44,45] by demonstrating the semi-independent stability of folding blocks of rRNA and r-proteins. In particular, since parts of domains IV and V can remain structured while the other unfolds, we can from the LiCl core particles derive two distinct pathways where either of these blocks independently remains folded ( Figure 6). ...
Ribosomes are complex ribonucleoprotein particles. Purified 50S ribosomes subjected to high-salt wash, removing a subset of ribosomal proteins (r-proteins), were shown as competent for in vitro assembly into functional 50S subunits. Here, we used cryo-EM to determine the structures of such LiCl core particles derived from E. coli 50S subunits. A wide range of complexes with large variations in the extent of the ordered 23S rRNA and the occupancy of r-proteins were resolved to between 2.8 Å and 9 Å resolution. Many of these particles showed high similarity to in vivo and in vitro assembly intermediates, supporting the inherent stability or metastability of these states. Similar to states in early ribosome assembly, the main class showed an ordered density for the particle base around the exit tunnel, with domain V and the 3′-half of domain IV disordered. In addition, smaller core particles were discovered, where either domain II or IV was unfolded. Our data support a multi-pathway in vitro disassembly process, similar but reverse to assembly. Dependencies between complex tertiary RNA structures and RNA-protein interactions were observed, where protein extensions dissociated before the globular domains. We observed the formation of a non-native RNA structure upon protein dissociation, demonstrating that r-proteins stabilize native RNA structures and prevent non-native interactions also after folding.
... 19 Siibak et al. isolated large subunit intermediates, accumulating in cells treated with chloramphenicol and erythromycin. 20 Both antibiotics produced two large subunit intermediates that migrated as particles with sedimentation coefficients of 35S and 45S. The extent of 10 Ψ-isomerizations and 13 nucleoside methylations on the 35S, 45S intermediates, and 50S large subunit was investigated using high-performance liquid chromatography and gel electrophoresis primer extension. ...
... The modifications that were largely missing from the 35S intermediates but were placed at the same extent in the 45S intermediates and the 50S large subunit were considered to occur during the intermediate stages of large subunit ribosome assembly. 20 Lastly, the modifications that were largely missing in the intermediates and present only in the 50S large subunit were considered to occur during the late stages of large subunit ribosome assembly. 20 More recently, Popova et al. and Rabuck-Gibbsons et al. isolated large subunit intermediates from wild-type cells and cells lacking the DEAD-box RNA helicase SrmB. ...
... 20 Lastly, the modifications that were largely missing in the intermediates and present only in the 50S large subunit were considered to occur during the late stages of large subunit ribosome assembly. 20 More recently, Popova et al. and Rabuck-Gibbsons et al. isolated large subunit intermediates from wild-type cells and cells lacking the DEAD-box RNA helicase SrmB. 21,22 Using quantitative mass spectrometry (MS), they determined the time points during large subunit assembly that the incorporations of four Ψ nucleotides, m 3 Ψ, 14 methylated nucleosides, and OH 5 C occur. ...
... The reconstructed series of particles subjected to r-protein dissociation corroborates the multi-pathway of assembly [13,44,45] by demonstrating the semi-independent stability of folding blocks of rRNA and r-proteins. In particular, since parts of domains IV and V can remain structured while the other unfolds, we can from the LiCl core particles derive two distinct pathways where either of these blocks independently remains folded ( Figure 6). ...
Ribosomes are complex ribonucleoprotein particles. Purified 50S ribosomes subjected to high-salt wash, removing a subset of ribosomal proteins (r-proteins), were early shown competent for in vitro assembly into functional 50S subunits. We here used cryo-EM to determine the structure of such LiCl core particles derived from E. coli 50S subunits. A wide range of complexes with large variation in extent of ordered 23S rRNA and occupancy of r-proteins could be identified, and resolved to between 2.8 Å and 9 Å resolution. Many of these particles showed high similarity to in vivo and in vitro assembly intermediates, supporting the inherent stability or metastability of these states. Similar to states in early ribosome assembly, the main class showed ordered density for 23S rRNA domains 0, I, II, III, VI and the 5’-half of domain IV. In addition, smaller core particles were discovered, which show that the most stable part of the 50S under high-salt conditions includes parts of domain 0 and most of domains I, III and the 5’-half of domain IV and four to eight r-proteins. Our data support a multi-pathway disassembly process based on independent folding blocks, similar but reverse to the assembly process. The study provides examples of dependencies between complex tertiary RNA structure and RNA-protein interactions where protein extensions dissociate before the globular domains. We observe formation of a non-native RNA structure upon protein dissociation, demonstrating that r-proteins stabilize native RNA structure and prevent non-native interactions also after folding.
IMPORTANCE
Ribosome assembly and stability remain only partially understood. Incubation of ribosomes with salts was early shown to induce dissociation of the more loosely bound ribosomal proteins (r-proteins) and formation of so-called core particles. In this work, cryo-EM imaging of 50S LiCl core particles from E. coli for the first time allowed structural characterization of such particles of different size. The smallest particles demonstrate what constitutes the smallest stable core of the 50S ribosomal subunit, and the sequential comparison with larger particles show how the ribosome disassembles and assembles in layers of rRNA structure stabilized by globular domains and extended tails of r-proteins. Major insights are that ribosomes disassemble along different paths, that dissociation of r-proteins can induce misfolding of rRNA and that extended tails of r-proteins dissociate from rRNA before the globular domains. The characterized particles can be used in future mechanistic studies of ribosome biogenesis.
