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.

The Bacillus subtilis ywbD gene encodes RlmQ, the 23S rRNA methyltransferase forming m7G2574 in the A-site of the peptidyl transferase center.

Ribosomal RNA contains many posttranscriptionally modified nucleosides, particularly in the functional parts of the ribosome. The distribution of these modifications varies from one organism to another. In Bacillus subtilis, the model organism for Gram-positive bacteria, mass spectrometry experiments revealed the presence of 7-methylguanosine (m7G) at position 2574 of the 23S rRNA, which lies in the A-site of the peptidyl transferase center of the large ribosomal subunit. Testing several m7G methyltransferase candidates allowed us to identify the RlmQ enzyme, encoded by the ywbD open reading frame, as the MTase responsible for this modification. The enzyme methylates free RNA and not ribosomal 50S or 70S particles, suggesting that modification occurs in the early steps of ribosome biogenesis.

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.

Interactions between terminal ribosomal RNA helices stabilize the E. coli large ribosomal subunit.

The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro. RNA tags in the Escherichia coli 50S subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that E. coli 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild-type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.

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.

Mirror-image T7 transcription of chirally inverted ribosomal and functional RNAs.

To synthesize a chirally inverted ribosome with the goal of building mirror-image biology systems requires the preparation of kilobase-long mirror-image ribosomal RNAs that make up the structural and catalytic core and about two-thirds of the molecular mass of the mirror-image ribosome. Here, we chemically synthesized a 100-kilodalton mirror-image T7 RNA polymerase, which enabled efficient and faithful transcription of the full-length mirror-image 5S, 16S, and 23S ribosomal RNAs from enzymatically assembled long mirror-image genes. We further exploited the versatile mirror-image T7 transcription system for practical applications such as biostable mirror-image riboswitch sensor, long-term storage of unprotected kilobase-long l-RNA in water, and l-ribozyme-catalyzed l-RNA polymerization to serve as a model system for basic RNA research.

The Bacillus subtilis open reading frame ysgA encodes the SPOUT methyltransferase RlmP forming 2'-O-methylguanosine at position 2553 in the A-loop of 23S rRNA.

A previous bioinformatic analysis predicted that the ysgA open reading frame of Bacillus subtilis encodes an RNA methyltransferase of the SPOUT superfamily. Here we show that YsgA is the 2'-O-methyltransferase that targets position G2553 (Escherichia coli numbering) of the A-loop of 23S rRNA. This was shown by a combination of biochemical and mass spectrometry approaches using both rRNA extracted from B. subtilis wild-type or ΔysgA cells and in vitro synthesized rRNA. When the target G2553 is mutated, YsgA is able to methylate the ribose of adenosine. However, it cannot methylate cytidine nor uridine. The enzyme modifies free 23S rRNA but not the fully assembled ribosome nor the 50S subunit, suggesting that the modification occurs early during ribosome biogenesis. Nevertheless, ribosome subunits assembly is unaffected in a B. subtilis ΔysgA mutant strain. The crystal structure of the recombinant YsgA protein, combined with mutagenesis data, outlined in this article highlights a typical SPOUT fold preceded by an L7Ae/L30 (eL8/eL30 in a new nomenclature) amino-terminal domain.

RNA Post-Transcriptional Modifications in Two Large Subunit Intermediates Populated in E. coli Cells Expressing Helicase Inactive R331A DbpA.

23S ribosomal RNA (rRNA) of Escherichia coli 50S large ribosome subunit contains 26 post-transcriptionally modified nucleosides. Here, we determine the extent of modifications in the 35S and 45S large subunit intermediates, accumulating in cells expressing the helicase inactive DbpA protein, R331A, and the native 50S large subunit. The modifications we characterized are 3-methylpseudouridine, 2-methyladenine, 5-hydroxycytidine, and nine pseudouridines. These modifications were detected using 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT) treatment followed by alkaline treatment. In addition, KMnO4 treatment of 23S rRNA was employed to detect 5-hydroxycytidine modification. CMCT and KMnO4 treatments produce chemical changes in modified nucleotides that cause reverse transcriptase misincorporations and deletions, which were detected employing next-generation sequencing. Our results show that the 2-methyladenine modification and seven uridines to pseudouridine isomerizations are present in both the 35S and 45S to similar extents as in the 50S. Hence, the enzymes that perform these modifications, namely, RluA, RluB, RluC, RluE, RluF, and RlmN, have already acted in the intermediates. Two uridines to pseudouridine isomerizations, the 3-methylpseudouridine and 5-hydroxycytidine modifications, are significantly less present in the 35S and 45S, as compared to the 50S. Therefore, the enzymes that incorporate these modifications, RluD, RlmH, and RlhA, are in the process of modifying the 35S and 45S or will incorporate these modifications during the later stages of ribosome assembly. Our study employs a novel high throughput and single nucleotide resolution technique for the detection of 2-methyladenine and two novel high throughput and single nucleotide resolution techniques for the detection of 5-hydroxycytidine.

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.

50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA.

Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2'-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a postcatalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144, which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes, even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.

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.

Expansion of the Genetic Code Through the Use of Modified Bacterial Ribosomes.

Biological protein synthesis is mediated by the ribosome, and employs ~20 proteinogenic amino acids as building blocks. Through the use of misacylated tRNAs, presently accessible by any of several strategies, it is now possible to employ in vitro and in vivo protein biosynthesis to elaborate proteins containing a much larger variety of amino acid building blocks. However, the incorporation of this broader variety of amino acids is limited to those species utilized by the ribosome. As a consequence, virtually all of the substrates utilized over time have been L-α-amino acids. In recent years, a variety of structural and biochemical studies have provided important insights into those regions of the 23S ribosomal RNA that are involved in peptide bond formation. Subsequent experiments, involving the randomization of key regions of 23S rRNA required for peptide bond formation, have afforded libraries of E. coli harboring plasmids with the rrnB gene modified in the key regions. Selections based on the use of modified puromycin derivatives with altered amino acids then identified clones uniquely sensitive to individual puromycin derivatives. These clones often recognized misacylated tRNAs containing altered amino acids similar to those in the modified puromycins, and incorporated the amino acid analogues into proteins. In this fashion, it has been possible to realize the synthesis of proteins containing D-amino acids, ß-amino acids, phosphorylated amino acids, as well as long chain and cyclic amino acids in which the nucleophilic amino group is not in the α-position. Of special interest have been dipeptides and dipeptidomimetics of diverse utility.

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.

Investigation of Allosteric Effect of 2,8-Dimethylation of A2503 in E. coli 23S rRNA by Molecular-Dynamics Simulations.

Ribosome is a molecular machine that synthesizes all cellular proteins. It also is a target of about half of the clinically used antibiotics. Adaptive chemical modification of ribosomal RNAs residues is one of the ways to provide resistance to certain antibiotics. A curious example of such modification is 2,8-dimethylation of A2503 in 23S rRNA, which induces resistance to phenols, linkosamides, oxazolidinones, pleuromutilins, and certain macrolides. In this article the effect of 2,8-dimethylation of A2503 on conformation and mobility of RNA residues of the 70S E. coli ribosome was investigated employing molecular dynamics simulations method. Significant alterations were detected both in the immediate environment of the 2503 23S rRNA residue and in the nucleotides located deeper in the nascent peptide exit tunnel (NPET), which are known to be involved in signal transmission from the antibiotics bound in the NPET to the peptidyl transferase center. These alterations shift the ribosome towards the A/A, P/P-state from the conformationally different state - P/P, E/E one in our case. The obtained results allow us to conclude that the effect of m2m8A2503 modification involves additional stabilization of the A/A, P/P-state favoring the peptidyl transferase reaction (PTR) contrary to antibiotics that inhibit PTR.

Incorporating a Thiophosphate Modification into a Common RNA Tetraloop Motif Causes an Unanticipated Stability Boost.

GNRA (N = A, C, G, or U; R = A or G) tetraloops are common RNA secondary structural motifs and feature a phosphate stacked atop a nucleobase. The rRNA sarcin/ricin loop (SRL) is capped by GApGA, and the phosphate p stacks on G. We recently found that regiospecific incorporation of a single dithiophosphate (PS2) but not a monothiophosphate (PSO) instead of phosphate in the backbone of RNA aptamers dramatically increases the binding affinity for their targets. In the RNA:thrombin complex, the key contribution to the 1000-fold tighter binding stems from an edge-on contact between PS2 and a phenylalanine ring. Here we investigated the consequences of replacing the SRL phosphate engaged in a face-on interaction with guanine with either PS2 or PSO for stability. We found that PS2···G and Rp-PSO···G contacts stabilize modified SRLs compared to the parent loop to unexpected levels: up to 6.3 °C in melting temperature Tm and -4.7 kcal/mol in ΔΔG°. Crystal structures demonstrate that the vertical distance to guanine for the closest sulfur is just 0.05 Å longer on average compared to that of oxygen despite the larger van der Waals radius of the former (1.80 Å for S vs 1.52 Å for O). The higher stability is enthalpy-based, and the negative charge as assessed by a neutral methylphosphonate modification plays only a minor role. Quantum mechanical/molecular mechanical calculations are supportive of favorable dispersion attraction interactions by sulfur making the dominant contribution. A stacking interaction between phosphate and guanine (SRL) or uracil (U-turn) is also found in newly classified RNA tetraloop families besides GNRA.

RNA sectors and allosteric function within the ribosome.

The ribosome translates the genetic code into proteins in all domains of life. Its size and complexity demand long-range interactions that regulate ribosome function. These interactions are largely unknown. Here, we apply a global coevolution method, statistical coupling analysis (SCA), to identify coevolving residue networks (sectors) within the 23S ribosomal RNA (rRNA) of the large ribosomal subunit. As in proteins, SCA reveals a hierarchical organization of evolutionary constraints with near-independent groups of nucleotides forming physically contiguous networks within the three-dimensional structure. Using a quantitative, continuous-culture-with-deep-sequencing assay, we confirm that the top two SCA-predicted sectors contribute to ribosome function. These sectors map to distinct ribosome activities, and their origins trace to phylogenetic divergences across all domains of life. These findings provide a foundation to map ribosome allostery, explore ribosome biogenesis, and engineer ribosomes for new functions. Despite differences in chemical structure, protein and RNA enzymes appear to share a common internal logic of interaction and assembly.