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.

Exploring a unique class of flavoenzymes: Identification and biochemical characterization of ribosomal RNA dihydrouridine synthase.

Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome's peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature's preference for utilizing reduced flavin in the reduction of uridines and their derivatives.

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.

Structural basis of Cfr-mediated antimicrobial resistance and mechanisms to evade it.

The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin.

An Atlas of the base inter-RNA stacks involved in bacterial translation.

Nucleobase-specific noncovalent interactions play a crucial role in translation. Herein, we provide a comprehensive analysis of the stacks between different RNA components in the crystal structures of the bacterial ribosome caught at different translation stages. Analysis of tRNA||rRNA stacks reveals distinct behaviour; both the A-and E-site tRNAs exhibit unique stacking patterns with 23S rRNA bases, while P-site tRNAs stack with 16S rRNA bases. Furthermore, E-site stacks exhibit diverse face orientations and ring topologies-rare for inter-chain RNA interactions-with higher average interaction energies than A or P-site stacks. This suggests that stacking may be essential for stabilizing tRNA progression through the E-site. Additionally, mRNA||rRNA stacks reveal other geometries, which depend on the tRNA binding site, whereas 16S rRNA||23S rRNA stacks highlight the importance of specific bases in maintaining the integrity of the translational complex by linking the two rRNAs. Furthermore, tRNA||mRNA stacks exhibit distinct geometries and energetics at the E-site, indicating their significance during tRNA translocation and elimination. Overall, both A and E-sites display a more diverse distribution of inter-RNA stacks compared to the P-site. Stacking interactions in the active ribosome are not simply accidental byproducts of biochemistry but are likely invoked to compensate and support the integrity and dynamics of translation.

Cryo-EM captures a unique conformational rearrangement in 23S rRNA helices of the Mycobacterium 50S subunit.

Structural investigations of the ribosomes isolated from pathogenic and non-pathogenic Mycobacterium species have identified several mycobacteria-specific structural features of ribosomal RNA and proteins. Here, we report structural evidence of a hitherto unknown conformational switch of mycobacterium 23S rRNA helices (H54a and H67-H71). Cryo-electron microscopy (cryo-EM) structures (~3-4 Å) of the M. smegmatis (Msm) log-phase 50S ribosomal subunit revealed conformational variability in H67-H71 region of the 23S rRNA, and manifested that, while H68 possesses the usual stretched conformation in one class of the maps, another one exhibits a bulge-out, fused density of H68-H69 at the inter-subunit surface, indicating an intrinsic dynamics of these rRNA helices. Remarkably, altered conformation of H68 forming a more prominent bulge-out structure at the inter-subunit surface of the 50S subunit due to the conformational rearrangements of 23S rRNA H67-H71 region was clearly visualized in a 3 Å cryo-EM map of the 50S subunit obtained from the stationary phase ribosome dataset. The Msm50S subunit having such bulge-out conformation at the intersubunit surface would be incompatible for associating with the 30S subunit due to its inability to form major inter-subunit bridges. Evidently, availability of active 70S ribosome pool can be modulated by stabilizing either one of the H68 conformation.

Comparing FRET Pairs that Report on Intersubunit Rotation in Bacterial Ribosomes.

Mediated by elongation factor G (EF-G), ribosome translocation along mRNA is accompanied by rotational movement between ribosomal subunits. Here, we reassess whether the intersubunit rotation requires GTP hydrolysis by EF-G or can occur spontaneously. To that end, we employ two independent FRET assays, which are based on labeling either ribosomal proteins (bS6 and bL9) or rRNAs (h44 of 16S and H101 of 23S rRNA). Both FRET pairs reveal three FRET states, corresponding to the non-rotated, rotated and semi-rotated conformations of the ribosome. Both FRET assays show that in the absence of EF-G, pre-translocation ribosomes containing deacylated P-site tRNA undergo spontaneous intersubunit rotations between non-rotated and rotated conformations. While the two FRET pairs exhibit largely similar behavior, they substantially differ in the fraction of ribosomes showing spontaneous fluctuations. Nevertheless, instead of being an invariable intrinsic property of each FRET pair, the fraction of spontaneously fluctuating molecules changes in both FRET assays depending on experimental conditions. Our results underscore importance of using multiple FRET pairs in studies of ribosome dynamics and highlight the role of thermally-driven large-scale ribosome rearrangements in translation.

Roles of the leader-trailer helix and antitermination complex in biogenesis of the 30S ribosomal subunit.

Ribosome biogenesis occurs co-transcriptionally and entails rRNA folding, ribosomal protein binding, rRNA processing, and rRNA modification. In most bacteria, the 16S, 23S and 5S rRNAs are co-transcribed, often with one or more tRNAs. Transcription involves a modified RNA polymerase, called the antitermination complex, which forms in response to cis-acting elements (boxB, boxA and boxC) in the nascent pre-rRNA. Sequences flanking the rRNAs are complementary and form long helices known as leader-trailer helices. Here, we employed an orthogonal translation system to interrogate the functional roles of these RNA elements in 30S subunit biogenesis in Escherichia coli. Mutations that disrupt the leader-trailer helix caused complete loss of translation activity, indicating that this helix is absolutely essential for active subunit formation in the cell. Mutations of boxA also reduced translation activity, but by only 2- to 3-fold, suggesting a smaller role for the antitermination complex. Similarly modest drops in activity were seen upon deletion of either or both of two leader helices, termed here hA and hB. Interestingly, subunits formed in the absence of these leader features exhibited defects in translational fidelity. These data suggest that the antitermination complex and precursor RNA elements help to ensure quality control during ribosome biogenesis.

Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome.

The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3'-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

Insights into the ribosome function from the structures of non-arrested ribosome-nascent chain complexes.

During protein synthesis, the growing polypeptide threads through the ribosomal exit tunnel and modulates ribosomal activity by itself or by sensing various small molecules, such as metabolites or antibiotics, appearing in the tunnel. While arrested ribosome-nascent chain complexes (RNCCs) have been extensively studied structurally, the lack of a simple procedure for the large-scale preparation of peptidyl-tRNAs, intermediates in polypeptide synthesis that carry the growing chain, means that little attention has been given to RNCCs representing functionally active states of the ribosome. Here we report the facile synthesis of stably linked peptidyl-tRNAs through a chemoenzymatic approach based on native chemical ligation and use them to determine several structures of RNCCs in the functional pre-attack state of the peptidyl transferase centre. These structures reveal that C-terminal parts of the growing peptides adopt the same uniform ß-strand conformation stabilized by an intricate network of hydrogen bonds with the universally conserved 23S rRNA nucleotides, and explain how the ribosome synthesizes growing peptides containing various sequences with comparable efficiencies.

Computationally-guided design and selection of high performing ribosomal active site mutants.

Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g. library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing of combined ribosomal assembly and translation activity, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design and selection may serve as a powerful tool for rRNA engineering.

VapC toxin switches M. smegmatis cells into dormancy through 23S rRNA cleavage.

Mycobacterium tuberculosis is an extremely successful pathogen known for its ability to cause latent infection. The latter is connected with the bacterium resting state development and is considered to be based on the activity of toxin-antitoxin (TA) systems at least in part. Here we studied the physiological and proteomic consequences of VapC toxin overexpression together with the features of the protein synthesis apparatus and compared them with the characteristics of dormant mycobacterial cells in an M. smegmatis model. The findings allow suggesting the mechanism mycobacteria enter dormancy, which is realized through VapC-caused cleavage of the 23S rRNA Sarcin-Ricin loop followed by conservation of stalled ribosomes in a membrane-associated manner. The found features of resting mycobacteria protein synthesis apparatus hypothesize the mechanisms of resuscitation from dormancy through the ribosomes de-association off the membrane accompanied by the 23S rRNA break curing, and could be of value for the development of principally new antituberculosis agents.

Structure of Escherichia coli heat shock protein Hsp15 in complex with the ribosomal 50S subunit bearing peptidyl-tRNA.

In Escherichia coli, the heat shock protein 15 (Hsp15) is part of the cellular response to elevated temperature. Hsp15 interacts with peptidyl-tRNA-50S complexes that arise upon dissociation of translating 70S ribosomes, and is proposed to facilitate their rescue and recycling. A previous structure of E. coli Hsp15 in complex with peptidyl-tRNA-50S complex reported a binding site located at the central protuberance of the 50S subunit. By contrast, recent structures of RqcP, the Hsp15 homolog in Bacillus subtilis, in complex with peptidyl-tRNA-50S complexes have revealed a distinct site positioned between the anticodon-stem-loop (ASL) of the P-site tRNA and H69 of the 23S rRNA. Here we demonstrate that exposure of E. coli cells to heat shock leads to a decrease in 70S ribosomes and accumulation of 50S subunits, thus identifying a natural substrate for Hsp15 binding. Additionally, we have determined a cryo-EM reconstruction of the Hsp15-50S-peptidyl-tRNA complex isolated from heat shocked E. coli cells, revealing that Hsp15 binds to the 50S-peptidyl-tRNA complex analogously to its B. subtilis homolog RqcP. Collectively, our findings support a model where Hsp15 stabilizes the peptidyl-tRNA in the P-site and thereby promotes access to the A-site for putative rescue factors to release the aberrant nascent polypeptide chain.

Structural Consequences of Deproteinating the 50S Ribosome.

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.

Targeted editing and evolution of engineered ribosomes in vivo by filtered editing.

Genome editing technologies introduce targeted chromosomal modifications in organisms yet are constrained by the inability to selectively modify repetitive genetic elements. Here we describe filtered editing, a genome editing method that embeds group 1 self-splicing introns into repetitive genetic elements to construct unique genetic addresses that can be selectively modified. We introduce intron-containing ribosomes into the E. coli genome and perform targeted modifications of these ribosomes using CRISPR/Cas9 and multiplex automated genome engineering. Self-splicing of introns post-transcription yields scarless RNA molecules, generating a complex library of targeted combinatorial variants. We use filtered editing to co-evolve the 16S rRNA to tune the ribosome's translational efficiency and the 23S rRNA to isolate antibiotic-resistant ribosome variants without interfering with native translation. This work sets the stage to engineer mutant ribosomes that polymerize abiological monomers with diverse chemistries and expands the scope of genome engineering for precise editing and evolution of repetitive DNA sequences.

Single gene targeted nanopore sequencing enables simultaneous identification and antimicrobial resistance detection of sexually transmitted infections.

OBJECTIVES: To develop a simple DNA sequencing test for simultaneous identification and antimicrobial resistance (AMR) detection of multiple sexually transmitted infections (STIs). METHODS: Real-time PCR (qPCR) was initially performed to identify Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) infections among a total of 200 vulvo-vaginal swab samples from female sex workers in Ecuador. qPCR positive samples plus qPCR negative controls for these STIs were subjected to single gene targeted PCR MinION-nanopore sequencing using the smartphone operated MinIT. RESULTS: Among 200 vulvo-vaginal swab samples 43 were qPCR positive for at least one of the STIs. Single gene targeted nanopore sequencing generally yielded higher pathogen specific read counts in qPCR positive samples than qPCR negative controls. Of the 26 CT, NG or MG infections identified by qPCR, 25 were clearly distinguishable from qPCR negative controls by read count. Discrimination of TV qPCR positives from qPCR negative controls was poorer as many had low pathogen loads (qPCR cycle threshold >35) which produced few specific reads. Real-time AMR profiling revealed that 3/3 NG samples identified had gyrA mutations associated with fluoroquinolone resistance, 2/10 of TV had mutations related to metronidazole resistance, while none of the MG samples possessed 23S rRNA gene mutations contributing to macrolide resistance. CONCLUSIONS: Single gene targeted nanopore sequencing for diagnosing and simultaneously identifying key antimicrobial resistance markers for four common genital STIs shows promise. Further work to optimise accuracy, reduce costs and improve speed may allow sustainable approaches for managing STIs and emerging AMR in resource poor and laboratory limited settings.

Dynamic 23S rRNA modification ho5C2501 benefits Escherichia coli under oxidative stress.

Post-transcriptional modifications are added to ribosomal RNAs (rRNAs) to govern ribosome biogenesis and to fine-tune protein biosynthesis. In Escherichia coli and related bacteria, RlhA uniquely catalyzes formation of a 5-hydroxycytidine (ho5C) at position 2501 of 23S rRNA. However, the molecular and biological functions as well as the regulation of ho5C2501 modification remain unclear. We measured growth curves with the modification-deficient ΔrlhA strain and quantified the extent of the modification during different conditions by mass spectrometry and reverse transcription. The levels of ho5C2501 in E. coli ribosomes turned out to be highly dynamic and growth phase-dependent, with the most effective hydroxylation yields observed in the stationary phase. We demonstrated a direct effect of ho5C2501 on translation efficiencies in vitro and in vivo. High ho5C2501 levels reduced protein biosynthesis which however turned out to be beneficial for E. coli for adapting to oxidative stress. This functional advantage was small under optimal conditions or during heat or cold shock, but becomes pronounced in the presence of hydrogen peroxide. Taken together, these data provided first functional insights into the role of this unique 23S rRNA modification for ribosome functions and bacterial growth under oxidative stress.

Maturation of 23S rRNA includes removal of helix H1 in many bacteria.

In most bacteria, the three ribosomal RNAs (rRNAs) are encoded together in each of several near-identical operons. As soon as the nascent precursor rRNA emerges from RNA polymerase, ribosome assembly begins. This process entails ribosomal protein binding, rRNA folding, rRNA modification, and rRNA processing. In the model organisms Escherichia coli and Bacillus subtilis, rRNA processing results in similar mature rRNAs, despite substantial differences in the cohort of RNAses involved. A recent study of Flavobacterium johnsoniae, a member of the phylum Bacteroidota (formerly Bacteroidetes), revealed that helix H1 of 23S rRNA is absent from ribosomes, apparently a consequence of rRNA maturation. In this work, we mined RNA-seq data from 19 individual organisms and ocean metatranscriptomic samples to compare rRNA processing across diverse bacterial lineages. We found that mature ribosomes from multiple clades lack H1, and typically these ribosomes also lack an encoded H98. For all groups analysed, H1 is predicted to form in precursor rRNA as part of a longer leader-trailer helix. Hence, we infer that evolutionary loss of H98 sets the stage for H1 removal during 50S subunit maturation.

Structural mechanism of GTPase-powered ribosome-tRNA movement.

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP-Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.

Structural basis for the activation of the DEAD-box RNA helicase DbpA by the nascent ribosome.

The adenosine triphosphate (ATP)-dependent DEAD-box RNA helicase DbpA from Escherichia coli functions in ribosome biogenesis. DbpA is targeted to the nascent 50S subunit by an ancillary, carboxyl-terminal RNA recognition motif (RRM) that specifically binds to hairpin 92 (HP92) of the 23S ribosomal RNA (rRNA). The interaction between HP92 and the RRM is required for the helicase activity of the RecA-like core domains of DbpA. Here, we elucidate the structural basis by which DbpA activity is endorsed when the enzyme interacts with the maturing ribosome. We used nuclear magnetic resonance (NMR) spectroscopy to show that the RRM and the carboxyl-terminal RecA-like domain tightly interact. This orients HP92 such that this RNA hairpin can form electrostatic interactions with a positively charged patch in the N-terminal RecA-like domain. Consequently, the enzyme can stably adopt the catalytically important, closed conformation. The substrate binding mode in this complex reveals that a region 5' to helix 90 in the maturing ribosome is specifically targeted by DbpA. Finally, our results indicate that the ribosome maturation defects induced by a dominant negative DbpA mutation are caused by a delayed dissociation of DbpA from the nascent ribosome. Taken together, our findings provide unique insights into the important regulatory mechanism that modulates the activity of DbpA.