Mechanistic insights into E. coli recovery from growth arrest

Domain: neuroscience
Published: 2026-04-11
Editorial outcome: minor_revision

Abstract
Bacteria survive hostile conditions by shutting down protein synthesis, but how they restart growth remains poorly understood. Here, we use an E. coli ΔrimM strain, which exhibits a prolonged growth arrest, as a model to investigate how bacteria recover from this state and restore protein synthesis. RimM is a conserved ribosome maturation factor for the 3'-major (head) domain of the 16S rRNA within the bacterial 30S subunit. The loss of RimM causes a longer delay in recovery than other 30S maturation factors, including RbfA. Cryo-EM analysis of ΔrimM ribosomes suggests a delayed recruitment of ribosomal proteins to the 30S head domain and increased occupancy of the initiation factors IF1 and IF3, as well as recruitment of the silencing factor RsfS to the 50S subunit. These coordinated changes provide a safeguarding mechanism to block the assembly of premature 70S ribosomes. Notably, while the delayed 30S assembly in ΔrimM reduces the activity of global protein synthesis during the recovery phase, bacteria attempt to compensate for this deficiency by producing higher levels of the ribosomal machinery, indicating a programmatic change in energy allocation. These findings highlight the importance of the RimM-assisted assembly of the ribosomal head domain for bacterial recovery from growth arrest.

Introduction
Bacteria arrest growth under stress conditions, many of which are relevant to clinical settings1. In growth arrest, bacteria terminate ribosome-mediated protein synthesis and dissociate ribosomes from the associated mRNA and tRNAs. Although bacteria have evolved a wide range of strategies to exit growth arrest2,3, one of the key elements is to re-assemble the ribosome to resume protein synthesis. However, the mechanisms by which bacterial ribosomes recover from growth arrest remain poorly understood, creating a critical gap in our knowledge of stress adaptation and posing a major challenge to develop effective treatments of bacterial infections. To address this gap, we use an E. coli ΔrimM strain, which exhibits a prolonged growth arrest4,5, as a model to identify factors that determine ribosome recovery.

RimM6 is an evolutionarily conserved ribosomal maturation factor for assembly of the bacterial 30S small ribosomal subunit. The assembly of the 30S begins with transcriptional synthesis of the 17S precursor of the 16S rRNA, which is then processed, post-transcriptionally modified, and folded into distinct structural domains7. The folding of the 16S rRNA occurs in coordination with maturation factors to guide the sequential recruitment8,9 of ribosomal proteins (r-proteins) to the 30S subunit. This process produces a series of pre-30S intermediates8-10 en route to the fully mature state of the 30S subunit.

Immature assembly of the 30S would produce error-prone ribosomes that disrupt the homeostasis of protein synthesis11,12. Based on the known maturation process of the 30S subunit9,10, ribosome binding factor A (RbfA) is considered to be the main organizer among the maturation factors of the 30S subunit. It is one of the first to engage in the 30S maturation pathway and it remains involved until the completion of maturation13,14, being released by the late-acting assembly factor RsgA15 and displaced by initiation factor IF314,16. RbfA is required for two important stages of maturation – formation of the central pseudoknot structure of the 16S rRNA in the early stage13, and the docking of helix 44 into the decoding site in a later stage17. In contrast, RimM comes after RbfA in the maturation process and has a shorter residence time on pre-30S particles18. It is specifically involved in proper folding of the 3'-major domain of the 16S rRNA localized in the 30S head domain5,13,19, where it recruits secondary- and tertiary-level r-proteins to stabilize assembly intermediates4,5,13,20.

Notably, RimM is not essential for bacterial survival, as several r-proteins (e.g., uS2, uS3, uS10, uS13, uS14, and uS19) can spontaneously bind to and facilitate the folding of the head region of the 16S rRNA4,5. However, E. coli strains lacking ΔrimM exhibited a markedly slow growth phenotype in previous studies4,5, although no direct comparisons of the growth recovery of ΔrimM with ΔrbfA have been reported, nor with E. coli strains lacking other 30S maturation factors and r-proteins. It is also unknown whether ΔrimM or the deletion of any 30S assembly factors induces coordinated response at the level of the large ribosomal subunit (50S), particularly through regulatory mechanisms that control subunit association21-23 rather than 50S maturation24 itself. One such mechanism involves the ribosomal silencing factor RsfS22, a conserved bacterial stress-response protein21. RsfS acts as an anti-association factor that sterically blocks 30S–50S joining, thereby downregulating translational activity as part of a general adaptive response to slow growth/stationary phase21,22,25. While the broad conservation of RsfS across bacterial species underscores its importance in regulating ribosome function21, how this stress-responsive factor adjusts its activity in response to the accumulation of pre-30S particles remains unknown. Although bacterial 30S intermediates lacking individual maturation factors have been reported4,13,26, including pre-30S particles deficient in RimM4,5, a comprehensive ex vivo structural view of 30S, 50S, and 70S particles from a ΔrimM strain is still lacking. This limits our understanding of how these particles coordinate to assemble ribosomes during the recovery from growth arrest.

Here, we show that an E. coli ΔrimM strain recovers from growth arrest markedly more slowly than isogenic ΔrbfA, ΔksgA (a late-stage maturation factor for the 30S decoding site)26, or ΔS9 (lacking r-protein uS9) strains. The ribosomes of the ΔrimM strain are specifically deficient in the translocation step of the elongation cycle, indicating deficiency in the 30S head domain27-29. This deficiency provides the basis for over-production of the ribosomal machinery across the lag and exponential phase. Cryo-EM analysis of ribosomal particles isolated from the early exponential phase of the ΔrimM strain revealed the persistent presence of IF1 and IF3 on the 30S subunit, delayed recruitment of r-proteins (uS3, uS10, uS13, uS14, uS19) to the head domain, as well as bound RsfS in both pre-50S and mature 50S particles. Consistent with the cryo-EM observations, proteomic analysis reveals upregulation of IF3 and RsfS, supporting the anti-association activity of each30 and reinforcing a ribosome quality control mechanism that prevents premature assembly of defective ribosomes. This large-scale ribosome quality control in the absence of RimM, which delays ribosome recovery to allow time to coordinate with multiple factors, highlights the importance of proper 3′-domain folding of the 16S rRNA and proper assembly of the head domain in the bacterial exit from growth arrest.

Results
An E. coli ΔrimM strain exhibits a pronounced delay in recovery from growth arrest
To identify the most critical factors for ribosome recovery from growth arrest, we compared a ΔrimM strain constructed in the E. coli K-12 MG1655 background with strains that lack viable checkpoints of 30S maturation. The ΔrbfA strain affects the maturation of the 16S rRNA 5′-end and decoding center14,18; the ΔksgA strain removes the terminal dimethylation checkpoint at the decoding site26; the ΔrsmE strain removes the methyl transferase that installs the m3U1498E modification in the 16S rRNA31 located in helix 44 (h44) near the A-site; the ΔS9 (ΔrpsI) strain alters a core r-protein that shapes the geometry of the head domain of the 30S subunit32,33; and the S13-mut (rpsM-mut) strain, which lacks the C-terminal residues (82–117) of the uS13 r-protein in the 30S head region, affects the formation of inter-subunit bridges with the 50S subunit34. Together these strains provide a spectrum to examine the 30S recovery pathway from early to late stages, enabling side-by-side comparisons.

An E. coli wild-type (wt) strain and a pair of isogenic ΔrimM and ΔrbfA strains were grown in LB to the stationary phase overnight (> 12 h), diluted into fresh LB, and the growth recovery was monitored by OD600 (Fig. 1a). While the wt strain had a lag phase of recovery of 1.1 h and ΔrbfA had a lag phase of 2.3 h, ΔrimM had a much longer lag phase of 4.5 h (Fig. 1a-c). The long lag phase of ΔrimM was striking, in contrast to the lag phase of ΔS9 (ΔrpsI; 1.1 h), S13-mut (rpsM-mut; 1.4 h), ΔksgA (1.6 h) and ΔrsmE (1.1 h) (Fig. 1d-f). The pronounced lag phase of ΔrimM relative to the wt was also observed after 5 overnights of growth arrest in LB (Supplementary Fig. 1a-c), and after one overnight of growth arrest in the minimal media M9 (Supplementary Fig. 1d-f) and in the defined media MOPS supplemented with glucose or galactose (Supplementary Fig. 1g-i). Additionally, as cells exited the lag phase and began the growth phase (OD600 of 0.2-0.3), the growth of ΔrimM remained slow relative to the wt and ΔS9 (Fig. 1d-f). Notably, the slow recovery of ΔrimM was not due to a loss of viability relative to the wt (Supplementary Fig. 1j), or due to the appearance of heterogeneity of cell populations (Supplementary Fig. 1k). However, ΔrimM exhibited a severe cold sensitivity (Supplementary Fig. 1l), consistent with the phenotypes that arise from a deficiency in ribosome assembly35,36.

Importantly, the slow recovery of ΔrimM from growth arrest was not rescued by the over-expression of rbfA, but by the over-expression of rimM itself; even over-expression of a variant of rimM (rimM-YY) was unable to fully rescue the slow recovery (Fig. 1g-i). The rimM-YY variant expressed a protein with reduced binding affinity to the 30S subunit, due to alanine substitutions of two highly conserved tyrosine residues (Y106A-Y107A) that affect RimM binding to the r-protein uS19 in the 30S head domain19. These results indicate that the slow recovery of ΔrimM is exclusively and specifically a consequence of rimM deficiency, rather than off-target effects, and that the high affinity of RimM binding to the 30S subunit is required to support efficient 30S maturation during recovery from growth arrest.

To determine how the absence of RimM affects ribosome recovery from growth arrest, we compared the cellular ribosome content between the wt and ΔrimM strains across the lag and exponential phase. Cells were grown to the stationary phase overnight (> 12 h), diluted into fresh LB, and sampled at OD600 of 0.1, 0.4, and 1.0, representing the recovery, early exponential, and late exponential phases, respectively. The ribosome content was measured using the ratio of total RNA over total protein ratio, which correlates with quantitative mass spectrometry measurements37. The results showed that ΔrimM produced higher ribosome content than the wt (Fig. 1j). This suggests an over-compensation effort of E. coli to produce more ribosomal subunits and ribosomes to address the deficiency of RimM.

ΔrimM ribosomes are defective in protein synthesis during the recovery phase
To identify the deficiency of ΔrimM ribosomes relative to wt ribosomes, we used kinetic assays to assess individual steps of the elongation cycle of protein synthesis. Rather than measuring the overall cellular rate of protein synthesis of a reporter gene37, we chose to gather kinetic information to identify the specific steps in an elongation cycle that manifest this ribosomal deficiency. We used our previously established reconstituted system, in which purified 70S ribosomes were programmed with an mRNA and supplied with tRNAs, initiation factors (IF1, IF2, IF3), elongation factors (EF-Tu, EF-G), and GTP29,38,39. To correlate with the data above showing ribosomal deficiency of ΔrimM, we purified 70S ribosomes from ΔrimM and wt cells harvested at OD600 of 0.1, 0.4, and 1.0, respectively.

We first assayed the dipeptide formation reaction, which evaluated the activity and quality of the ribosome in the A site. An initiation complex containing fMet-tRNAfMet in the P site and an empty A site with a CGU codon was supplied with a ternary complex of EF-Tu, GTP and Arg-tRNAArg. After GTP hydrolysis and Arg-tRNAArg accommodation, the ribosome synthesized dipeptide fMet-Arg in the A site (Fig. 2a). Using 35S-Met in the initiation complex, we measured dipeptide formation by electrophoretic TLC29,38,39. The relative plateau reflected the fraction of active ΔrimM ribosomes relative to wt ribosomes, while the relative kobs reported the rate constant of each relative to each other. The results showed that ΔrimM ribosomes were deficient relative to wt ribosomes across the selected phases of growth, exhibiting active fractions at 70-80% and rate constants at 60-70% (Fig. 2b, c and Supplementary Fig. 2a), indicating a defect in the assembly of the A site.

We next assayed tripeptide formation in the A site, where a dipeptide was synthesized first, followed by translocation of the deacylated tRNAfMet and the dipeptidyl 35S-Met-Arg-tRNAArg, after which Ser-tRNASer entered the A site, leading to synthesis of the tripeptide 35S-fMet-Arg-Ser (Fig. 2a). Kinetic parameters of this tripeptide formation were composite terms, including dipeptide formation in the A site, translocation of the dipeptidyl-tRNA from the A to the P site, and tripeptide formation in the A site. Since the kobs of tripeptide formation of both the ΔrimM and wt ribosomes was consistently lower than the respective dipeptide formation (Supplementary Fig. 2), we concluded that the composite terms of tripeptide formation were rate-limited by the event of translocation. Notably, while the kobs of ΔrimM ribosomes was similar relative to the respective wt ribosomes (Fig. 2e), the active fraction of ΔrimM ribosomes was particularly low during the recovery phase (Fig. 2d), indicating a specific deficiency of ΔrimM ribosomes in the translocation event. As the quality and efficiency of translocation is critically dependent on the 30S head domain27-29, the deficiency of ΔrimM ribosomes in the recovery phase is consistent with an immaturely assembled head domain. This provides a kinetic basis for ribosome deficiency, particularly the deficiency of the head domain, for the slow recovery of E. coli from growth arrest in the absence of RimM.

IF1 and IF3 safeguard the 30S head domain maturation in ΔrimM strain
To uncover the structural basis for this functional defect (Fig. 2d), we examined ribosomal particles from the ΔrimM strain by single-particle cryo-EM, using E. coli ΔrimM cells collected at OD600 = 0.4 (early exponential phase). For these experiments, we used the E. coli ΔrimM strain (MRE600 background), which exhibits reduced RNase activity40 and is well suited for preserving pre-30S maturation intermediates. Ribosomes were purified using high-salt sucrose gradient fractionation to preserve maturation intermediates and retain stably bound auxiliary factors. Importantly, this strain shows the same prolonged recovery phenotype as the MG1655 background and displays comparable ribosome profiles, including an over-representation of free subunits relative to 70S ribosomes, consistent with ongoing 30S maturation and delayed 70S assembly (Supplementary Fig. 3). To ensure that the ribosome population (OD600 = 0.4) is representative of other growth stages, we extended polysome profiling to ΔrimM cells collected at OD600 = 0.1 and 1.0, and stationary phase. Ribosome profiles at OD600 0.1 and 1.0 closely resembled those observed at OD600 0.4 (Supplementary Figs. 3, 4), indicating that the ribosomal phenotype analyzed by cryo-EM reflects a stable feature of ΔrimM cells during recovery and exponential growth. By comparison, during the stationary phase ΔrimM cells exhibited a distinct redistribution of ribosomal species relative to wt cells, including reduced formation of hibernating 100S ribosomes and an imbalance between free 30S and 50S subunits (Supplementary Fig. 4).

The cryo-EM analysis of the 30SΔrimM revealed a large fraction of immature 30S particles in which the head domain was poorly resolved or largely absent due to its immature, highly dynamic nature (Supplementary Fig. 5), consistent with previously described intermediates in 30S biogenesis4,5. Importantly, we identified three maturation states of the 30S subunit (I-III), all without the presence of the mRNA but bound with initiation factors IF1 and IF3 at their canonical positions near the 30S platform and A site41,42, respectively (Fig. 3 and Supplementary Figs. 6a, 7). These three states represent progressive stages of head domain formation, according to the Nomura assembly map43,44 for the stepwise incorporation of r-proteins into the 30S head domain and the concurrent stabilization of the 3′-major domain of the 16S rRNA (Fig. 3).

In state I, densities for r-proteins uS3, uS10, uS13, uS14 and uS19 were absent, and the 30S head domain displayed high conformational flexibility. This flexibility was especially pronounced in the 3′-major domain of the 16S rRNA, which normally depends on these r-proteins for stabilization. According to the Nomura assembly map43,44, uS13 and uS19 are secondary assembly proteins, while uS3, uS10, and uS14 are part of the tertiary (late) assembly stage. Structural analysis of the 3′-major domain of the 16S rRNA showed disordered regions in several key helices, including nucleotides 952–953 (helix h31), 959–969 (h31–32 junction), 983–989 (h32), 1049–1054 (h33), 1195–1201 and 1218–1225 (h38), 1230–1231 (h39), 1298–1302 (h41), 1307–1309 (h42), and 1330–1339 (h43) (Fig. 3a-c, top row). These helices form the structural core of the 30S head and are essential for inter-subunit bridge formation and the accommodation of tRNAs45,46. The absence of uS3, uS10, uS13, uS14, and uS19 correlated with destabilization in these regions, reinforcing the critical roles of these r-proteins in structuring the head domain during 30S maturation5,47. Therefore, 30SΔrimM state I represents an early intermediate in the maturation of the head domain in the absence of RimM.

In 30SΔrimM state II (Fig. 3a-c, middle row), r-proteins uS13 and uS19 (secondary assembly proteins) were bound to the 30S head domain. Their incorporation led to localized stabilization of the 3′-major domain of the 16S rRNA, showing partial compaction and structural ordering of the head domain as it progressed toward full maturation. This stabilization was particularly evident in rRNA helices h41, h42, and h43, which directly interacted with uS13 and uS19. However, several regions of the 16S rRNA within the head domain remained flexible, especially rRNA helices encompassing nucleotides 972–975 (h31), 1053–1056 (h33), and 1200–1205 (h38). Proper folding and stabilization of these regions would depend on the subsequent incorporation of tertiary assembly r-proteins, particularly uS3, uS10, and uS14.

In 30SΔrimM state III (Fig. 3a-c, bottom row), the late-assembly r-proteins uS3, uS10, and uS14 were fully incorporated into the 30S head domain. The 3′-major domain of the 16S rRNA showed ordered structures in regions that were previously dynamic or unfolded, indicating that structural maturation of the head domain was complete. Structural comparison with the mature 30S subunit42 confirmed that state III closely matches the native configuration in both r-protein composition and 16S rRNA folding (Supplementary Fig. 7), whereas states I and II exhibited progressive improvement in these features. IF1 and IF3 remained bound throughout all three states, indicating that their persistent presence plays a regulatory role30,48 to stabilize immature 30S subunits and to guide their stepwise maturation. This ensures that only properly assembled 30S subunits proceed to association with the 50S subunit to assemble functional 70S ribosomes for translation.

RsfS controls the 50S subunit association with 30S subunit and the 70S assembly
To test whether the deficiency of 30S assembly in the ΔrimM strain was coordinated with a regulatory mechanism that controls 50S subunit association, we performed single-particle cryo-EM on 50S subunits isolated from the E. coli ΔrimM strain at an OD of 0.4. The analysis revealed two major populations of 50S particles (Supplementary Fig. 6b and 8). One population consisted of immature 50S (pre-50S) subunits lacking helix 68 (-H68), a late-assembling helix of the 23S rRNA that is inserted between the L1 stalk and the central protuberance during the final stages of 50S maturation24,49,50. The second population consisted of fully mature 50S subunits with helix H68 correctly folded and integrated in its canonical position. Notably, some fractions of the pre-50S and 50S particles were bound with the ribosome silencing factor RsfSΔrimM (Fig. 4). Cryo-EM maps clearly resolved RsfS at its canonical binding site on the inter-subunit face of the 50S subunit, similar to previous structural studies of the 50S subunit isolated from S. aureus22 (Supplementary Fig. 9) and M. tuberculosis51. RsfS adopted a compact α/β fold and was in direct contact with r-protein uL14 near the L11 stalk. Its binding site formed a stable cleft between uL14 and adjacent 23S rRNA helices, which was supported by a dense network of electrostatic and hydrophobic interactions (Fig. 4). RsfS remained stably bound at this site regardless of the presence or absence of helix H68, which is located distantly on the opposite side of the 50S subunit near the L1 stalk (Fig. 4). These findings indicate that RsfS can play a dual role, acting as a maturation checkpoint that senses the late biogenesis state of the 50S subunit52, and as an anti-association factor that prevents premature 70S assembly22.

IF1, IF3 and RsfS control ribosomal maturation and association of subunits
The occupancy of IF1, IF3 and RsfS in ΔrimM ribosomal samples suggested an important role of each in the ribosomal maturation and 70S assembly process. To directly assess the importance of each, we performed a quantitative comparison of ribosomal samples from ΔrimM relative to the wt strain (MRE600) collected at OD of 0.4 (Supplementary Figs. 5, 6, 8, 10-14). This comparison showed that, in mature particles, the ribosomes of the two strains were structurally indistinguishable, whereas in immature particles, the two strains differed substantially in the occupancy of the factors, particularly in the 30S and 50S subunits (Fig. 5).

In the wt strain, fully assembled head domains of 30S subunits were found without incomplete intermediates. The only IF1/IF3-bound 30S class corresponded to a fully mature subunit (Fig. 5a), matching 30S state III, in which the 16S rRNA folding, r-protein placement, and IF1/IF3 binding sites were fully in matured state in both the wt and ΔrimM strains. Key structural domains – including the platform, body, and head – were intact, and the decoding center, comprising helices h44, h45, were fully ordered in both strains. This suggests that, once the head domain maturation is complete, 30SΔrimM subunits can support translation as efficiently as wt subunits.

In contrast, in the ΔrimM strain, immature 30S particles represented by 30SΔrimM states I and II, were accumulated as shown by immature head domains lacking late-binding r-proteins while retaining IF1 and IF3 (Fig. 5e). Notably, the disordered head domain structures agree with those of earlier cryo-EM studies collected from an rimM-depleted strain4,5. Quantitative analysis of the 30S subunits revealed (Fig. 5e) that IF1 and IF3 are more abundant in the immature 30S particles (30S I and II) compared to the wt. Complementary mass spectrometry showed elevated IF3 levels, while IF1 remained unchanged in the ΔrimM strain during the early exponential phase, suggesting that IF3 upregulation is a key regulatory mechanism in response to the absence of RimM (Supplementary Fig. 15).

The comparison of 50S subunits between the wt and ΔrimM strains revealed no significant differences in the 23S rRNA core or r-protein composition (Fig. 5b, c). Quantitative analysis of 50S particle populations (Fig. 5f) showed that in the wt strain, most 50S subunits were either fully mature or in a late-stage assembly intermediate lacking helix 68 (-H68) of the 23S rRNA. Only a small fraction of these particles was bound by RsfS. In contrast, the ΔrimM strain exhibited a higher proportion of mature 50S subunits, possibly a response to defective 30S assembly by overproducing the 50S subunits (Fig. 1j and Supplementary Fig. 3c). Notably, the number of RsfS-bound 50S particles was elevated in ΔrimM, including both pre-50S and mature 50S subunits (Fig. 5f). Additionally, mass spectrometry analysis also revealed elevated RsfS levels in the ΔrimM strain during early exponential growth, indicating that RsfS upregulation constitutes an adaptive stress response to impaired 30S biogenesis (Supplementary Fig. 15). Notably, the ΔrsfS deletion did not alleviate the prolonged recovery phase observed in the ΔrimM strain (Fig. 5g-i), likely suggesting that the high occupancy of IF1 and IF3 that remained bound to the 30S particles (Fig. 5e) was sufficient to prevent subunit joining.

Moreover, we found that 50S subunits of both the ΔrimM and wt strains contained a second copy of the r-protein bS20. While bS20 is typically located on the body of the 30S subunit, this second copy was observed in the 50S subunit, positioned near helices H4, H18, and H28, where it formed close contacts with the r-protein uL4 (Supplementary Fig. 16a, b). Notably, a recent cryo-EM analysis of P. urativorans ribosomes detected bS20 on both small and large subunits53, where the position in the large subunit matched that of our 50S maps. This second copy appeared to be a neutral and heterogeneous feature with no clear functional role53.

Despite the pronounced differences between the ΔrimM and wt strains either in the composition or maturation state of 30S and 50S subunits, the 70S ribosomes were identical (Fig. 5d). Structural comparisons revealed no detectable differences between the assembled ribosomes. In both cases, mature 30S and 50S subunits were incorporated without any rRNA folding defects, misalignment of inter-subunit bridges, or changes in the positioning of r-proteins. The only notable addition in the 70S ribosomes was the presence of a second copy of the r-protein bS20 in both strains (Supplementary Fig. 16c, d), similar to that observed in isolated 50S subunits. Both the 30S decoding center and the 50S peptidyl transferase center were correctly formed. Thus, while 30S subunits of ΔrimM are delayed in maturation, once they are matured, they are readily assembled with mature 50S subunits into 70S ribosomes that have the normal structural architecture.

Methods
Strain constructions
Isogenic E. coli K-12 MG1655 strains of ΔrimM, ΔrbfA and ΔksgA carrying a kanamycin resistance marker (kanR) were obtained from the Keio collection66 and infected into MG1655 recipients. MG1655 strains ΔrsfS and ΔrsmE were constructed using the λ-Red recombinase system67. A kanamycin resistance marker flanked by sequences homologous to the 5′ and 3′ ends of the chromosomal rsfS gene and rsmE gene were PCR amplified from a pKD4 plasmid using the primers described in Supplementary Table 1. The PCR amplified sequences were electroporated into E. coli MG1655 cells carrying the λ-Red recombinase expression plasmid pKD46. Kanamycin-resistant colonies were screened for their correct insertion to the rsfS locus and to the rsmE locus and were confirmed by PCR using the primers described in Supplementary Table 1. Each gene knockout locus was transferred to the E. coli MG1655 strain by P1 transduction using P1 phage lysates prepared from the corresponding knockout strains. The P1 phage lysate prepared from MG1655 ΔrimM was used to generate the MG1655 ΔrimM/ΔrsfS and MRE600 ΔrimM strains. All MG1655 strains were λDE3-lysogenized to enable protein expression from T7 expression vectors with a λDE3 Lysogenization Kit (#69734-M, Novagen). The K-12 strains ΔrpsI (lacking the uS9 protein) and rpsM-mut (encoding a mutant of uS13 lacking residues 82-117) were obtained from Kurt Fredrick68.

Plasmid constructions
A plasmid expressing His-rimM, codon-optimized for E. coli, was kindly provided by Dr. Ortega at McGill University. To generate a plasmid expressing His-rimM-Y106A/Y107A, site-directed mutagenesis was performed by inverse PCR with primers containing the mutation described in Supplementary Table 2, using pET15b-His-rimM as the template. To construct plasmids expressing rbfA, the rbfA gene was amplified from E. coli MG1655 genomic DNA using the primers described in Supplementary Table 2 and cloned into the pET15b vector by restriction enzyme digestion with NcoI and BamHI and ligation with T4 DNA ligase (#M0202, NEB). All constructs were confirmed by Sanger sequencing. An empty vector pET22b was used as a control.

Growth assay
Cells were inoculated into fresh LB medium, MOPS-rich medium (#M2105, Teknova), or M9 minimal medium at a 1:100 dilution after 1-day or 5-day pre-culture in LB and incubated in 24-well plates at 37ºC. Strains carrying a kanamycin resistance marker were grown in media supplemented with kanamycin. OD600 was measured over time in an Infinite M200 PRO plate reader (Tecan). MOPS medium was supplemented with glucose (#G0520, Teknova) or with galactose at 0.2% (w/v). The measured OD600 was fitted to the logarithmic growth equation:
N(t) = K / (1+e−r(t−tmid))
where N(t) is the OD600 at time t, K is the carrying capacity (i.e., maximum OD600), r is the intrinsic growth rate, and tmid is the time when the OD600 reaches half of K. Although the standard logarithmic equation uses the initial OD600 value (N0) as a parameter69, in this study N0 was used, as it had a better fit to the experimental data. Fitting was performed using non-linear least squares with Python v3.7.12 (scipy.optimize.curve_fit). OD600 values were baseline-corrected by subtracting the absorbance of the medium-only control. Lag time was defined as the time at which the OD600 reaches 0.2, which was calculated using the equation:
Lag time = tmid - ln(K/0.2 - 1) / r
The number of generations per hour was calculated using the equation:
#Generation = r / ln2

Ribosome content
Ribosome content was defined as the ratio of [total RNA]/[total protein] for a culture of cells37. For total protein quantification, cell cultures of 15 mL, 5 mL, and 2 mL grown at 37ºC were pelleted at OD600 of 0.1, 0.4, and 1.0, respectively. Cells were washed with water, resuspended in 0.2 mL of water, and flash frozen in liquid nitrogen. The suspension was thawed, lysed with 0.1 mL of 3 M NaOH at 100ºC for 5 min, and cooled to room temperature. Protein yield was determined by Bradford assay using Protein Assay Dye Reagent Concentrate (#5000006, Bio-Rad) and measured at 595 nm in an Infinite M200 PRO plate reader (Tecan). For total RNA quantification, the same volumes of culture as for protein quantification were pelleted and flash-frozen in liquid nitrogen. Total RNA was extracted using TRIzol reagent (#15596018, Invitrogen) according to the manufacturer’s instructions and quantified by OD260 reading in a Nanodrop spectrophotometer.

Viability assay
MG1655 strains wt and ΔrimM were cultured in LB at 37ºC for either 1 day or 5 days. To normalize the cell number, both cultures were first diluted to OD600 1.0 with fresh LB, then further diluted 105-fold. A 10 µL aliquot of the diluted culture was spread on LB plates and incubated at 37ºC until colonies appeared. Cell counts were calculated from the colony number within six areas of the same size on each plate. To assess whether OD600 provides a reliable proxy for cell number, wt and ΔrimM cultures were adjusted to the same OD600, serially diluted, and plated on LB agar. Colony-forming units (CFUs) were then compared between strains at identical dilutions to verify that equivalent OD600 values correspond to comparable numbers of viable cells.

Cold-sensitivity assay
MG1655 strains wt and ΔrimM were cultured in LB at 37ºC overnight, diluted 1:100 into fresh M9 minimal medium, and grown at 37ºC until OD600 reached 0.3-0.4 in the exponential phase. Ten-fold serial dilutions (100–10-5) were prepared and spotted on M9 plates, which were then incubated at either 37ºC or room temperature for 1 to 4 days.

Ribosome purification for in vitro translation assay
Tight-coupled 70S ribosomes were isolated from E. coli wt MRE600 and ΔrimM (rif+kan) MRE600 cells70. Cells were grown in LB medium, harvested at OD600 of 0.1, 0.4, and 1.0, flash frozen in liquid nitrogen and stored at -80°C. For ribosome preparation, frozen cells were thawed, washed once in a resuspension buffer (20 mM Tris-HCl, pH 7.5, 30 mM NH4Cl, 6 mM Mg(OAc)2 and 4 mM βME) and collected by centrifugation at 10,000 rpm at 4°C for 15 min in a SS-34 rotor (Sorvall). The cell pellet was resuspended in fresh resuspension buffer and lysed by three to five passes through a cell disruptor (EmulsiFlex C3, Avestin) at 5,000 psi. Cell debris was removed by two rounds of centrifugation at 16,000 rpm for 30 min at 4°C in a SS-34 rotor.

Ribosomes in the clarified supernatant were pelleted through an equal volume of 1.1 M sucrose cushion in buffer A (20 mM Tris-HCl, pH 7.5, 500 mM NH4Cl, 10.5 mM Mg(OAc)2, 4 mM βME, and 0.5 mM EDTA) by ultracentrifugation at 35,000 rpm for 20 h at 4°C in a Ti-70 rotor (Beckman). The crude ribosomal pellet was resuspended in fresh buffer A and subjected to a second round of pelleting through a sucrose cushion. The pelleted ribosomes were resuspended in buffer B (20 mM Tris-HCl, pH 7.5, 60 mM NH4Cl, 10.5 mM Mg(OAc)2, 4 mM βME and 0.5 mM EDTA) and separated on a 10-30% sucrose gradient in the same buffer. Gradients were centrifuged at 18,000 rpm for 18 h at 4°C in a SW28-Ti rotor (Beckman). The 70S peak was collected, pooled, and pelleted at 23,000 rpm for more than 24 h at 4°C in a 70Ti rotor. The resulting ribosome pellet was resuspended in buffer B, clarified by centrifugation at 13,500 rpm for 15 min at 4°C in a FA-45-30-11 rotor (Eppendorf), aliquoted, and flash frozen in liquid nitrogen. The ribosome concentration was determined by OD260, using 24 pmol per A260 unit as the conversion factor.

In vitro translation assays
Di- and tripeptide formation assays were performed using the codon-walk approach as previously described29,39 in a buffer (50 mM Tris-HCl, pH 7.5, 70 mM NH4Cl, 30 mM KCl, 3.5 mM MgCl2, 1 mM DTT, 0.5 mM spermidine) at 20°C unless otherwise indicated. The three tRNAs used in these experiments were over-expressed in E. coli and isolated as part of a total tRNA pool. Each elongator tRNA was separately charged with its cognate aminoacyl-tRNA synthetase. The initiator tRNAfMet was charged with [35S]-methionine and formylated with the methyl donor 10-formyltetrahydro-folate by adding formyl transferase to the aminoacylation reaction. All tRNAs were stored in 25 mM NaOAc (pH 5.0) at –70ºC. The mRNA template was prepared by in vitro transcription and contained a Shine-Dalgarno sequence and an AUG start site: 5’GGGAAGGAGGUAAAAAUGCGUUCGAAG(CCA)10, where the first three codons are underlined.

To monitor peptide bond formation, a 70S initiation complex was formed by incubating 70S ribosomes, fMet-tRNAfMet, mRNA, and initiation factors IF1, IF2, and IF3 in the HiFi buffer (50 mM Tris-HCl, pH 7.5, 70 mM NH4Cl, 30 mM KCl, 3.5 mM MgCl2, 1 mM DTT, 0.5 mM spermidine, and 1 mM GTP) for 25 min at 37ºC. For dipeptide formation, the ternary complex of Arg-tRNAArg was mixed with the initiation complex, while for tripeptide formation, both the ternary complex of Arg-tRNAArg and of Ser-tRNASer were mixed with the initiation complex at the same time. Each ternary complex was formed by first incubating EF-Tu in the HiFi buffer for 15 min at 37ºC, followed by 15 min in an ice bath after adding a charged tRNA. The complete reaction was formed by rapidly mixing these two solutions in a Kintek model RQF-3 chemical quench apparatus at 20ºC. Assays for dipeptide formation contained 0.4 µM 70S ribosome, 0.5 µM mRNA, 0.5 µM [35S]-fMet-tRNAfMet, 0.5 µM each IF1, IF2, and IF3, 0.5 µM Arg-tRNAArg (anticodon ACG, where A is modified to I (inosine) in cells), and 0.75 µM EF-Tu in the HiFi buffer. Assays for tripeptide formation additionally contained 0.5 µM EF-G, 0.5 µM Ser-tRNASer (anticodon GCU), and 1.5 µM EF-Tu. Timed aliquots from each reaction were quenched with 170 mM KOH and the tRNA-peptidyl linkages were hydrolyzed by incubation at 37ºC for at least 15 min. Aliquots of 0.65 µL were spotted onto a cellulose-backed plastic TLC sheet and electrophoresed at 1000 V in PYRAC buffer (62 mM pyridine, 3.48 M acetic acid, pH 2.7) until the marker dye bromophenol blue reached the water-oil interface at the anode29,39. The position of the origin was adjusted to maximize separation of the expected oligopeptide products. The separation of unreacted [35S]-fMet and each of the [35S]-fMet-peptide products was visualized by phosphorimaging and quantified using ImageQuant (GE Healthcare), and kinetic plots were fitted using Kaleidagraph (Synergy software).

Polysome profiling
MG1655 and MRE600 wt and ΔrimM strains were cultured in LB at 37ºC overnight, diluted 1:100 into fresh LB, and grown at 37ºC until OD600 reached 0.1, 0.4, 1.0 and stationary phase. The ΔrimM strains carrying a kanamycin resistance marker were grown in LB supplemented with kanamycin. Cell cultures of 50 mL were collected and centrifuged at 7,000 rpm for 10 min at 4ºC, and the pellets were frozen in liquid nitrogen. Pellets were thawed and resuspended in 650 µL of lysis buffer (25 mM HEPES, pH 7.5, 100 mM KOAc, 15 mM Mg(OAc)2, 1 mM DTT)71 supplemented with 6.5 µL of DNase I (#M0303L, NEB) or 0.01% n-Dodecyl-Beta-D-Maltoside (#850520, Avanti Research). Cells were lysed with approximately 400 mg of 0.1 mm Zirconia/Silica Beads (#11079101z, BioSpec) in at FastPrep-24 5G bead beating (#116005500, MP Biomedicals) instrument at 6.0-6.5 m/sec for 30-60 sec, repeated 3 times. Cell lysates were cleared by centrifugation at 13,000 rpm for 10 min at 4ºC in a refrigerated microcentrifuge (#C2500-R, Labnet Prism). The RNA concentrations were determined by OD260 in a Nanodrop spectrophotometer. Then 15-25 OD260 units of cell lysates were loaded onto a 10-40% sucrose gradient prepared in the lysis buffer. Ribosomes were fractionated by ultracentrifugation at 36,600 rpm for 3.5 hours at 4ºC in a SW-41 Ti rotor (Beckman), and the gradient profiles were recorded with a Gradient Station Fractionator (Biocomp).

LC-MS/MS analysis
Cells were grown under the same conditions as those used for cryo-EM sample preparation. LC–MS/MS analysis was conducted on two E. coli strains, MG1655 and MRE600. For each strain, both wt and ΔrimM variants were analyzed in biological triplicates. All raw data files were processed together in a single database search. Because the cryo-EM experiments were performed using the MRE600 strain, the present study focuses on the corresponding MRE600 proteomic dataset to maintain methodological consistency between the structural and proteomic and structural analyses.

For each biological replicate, 45 mL of culture was harvested by centrifugation at 5,000 rpm for 45 minutes at 4°C (JLA 8.1000 rotor). Protein samples from E. coli cell pellets of MRE600 wt and ΔrimM (rif+kan) strains (in 3 biological replicates) were extracted in SDT buffer (100 mM Tris-HCl, pH 7.6, 4% SDS, 100 mM DTT) in a thermomixer (Eppendorf ThermoMixer, 15min, 95°C, 750 rpm). After that, all samples were centrifuged (15 min, 20,000 x g) and the supernatants (~75 μg of total protein) were used for filter-aided sample preparation (FASP) as previously described72 using 0.75 μg of trypsin (sequencing grade; Sigma-Aldrich). The resulting peptides were extracted into LC-MS vials with 2.5% formic acid (FA) in 50% acetonitrile (ACN) and 100% ACN with the addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator (Thermo Fisher Scientific).

LC-MS/MS analyses of all peptides were done using the UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) connected to timsTOF HT mass spectrometer (Bruker). Before LC separation, tryptic digests were online concentrated and desalted using a trapping column (Acclaim PepMap Neo C18, dimensions 300 μm ID, 5 mm long, 5 μm particles, 100A, Thermo Fisher Scientific). The trapping column was then washed with 0.1% TFA and the peptides were eluted in backflush mode from the trapping column onto an analytical column (Aurora C18, 75μm ID, 250 mm long, 1.7 μm particles, Ion Opticks) over 90 min in a gradient program (flow rate 200 nL.min-1, 3-42% of mobile phase B; mobile phase A: 0.1% FA in water; mobile phase B: 0.1% FA in 80% ACN), followed by a system wash using 80% of mobile phase B. Equilibration of the trapping column and the analytical column was done before sample injection to a sample loop. The iRT peptide solution was automatically spiked into the sample by the LC autosampler during the sample loading phase. The analytical column was installed into a Captive Spray ion source (Bruker; temperatures set to 150 ºC) according to the manufacturer's instructions. Spray voltage and dry gas were set to 1.4kV and 3 L.min-1, respectively.

MS data were acquired in data-independent acquisition (DIA) mode with the base method m/z range of 100-1700 and 1/k0 range of 0.6-1.4 V×s×cm-2. The enclosed diaParameters.txt (attached as source data file) file defined a m/z 400-1000 precursor range with an equal windows size of 26 Th using two steps for each PASEF scan and a cycle time of 100 ms locked to 100% duty cycle. DIA data were processed in DIA-NN (version 2.1.0)73 in library-free mode against the iRT+trypsin database (12 sequences in total, version 2024/10), the common contaminant database cRAP (based on http://www.thegpm.org/crap; 111 sequences in total), and the protein database for Escherichia coli (https://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/reference_proteomes/Bacteria/UP000000625_83333.fasta.gz, version 2025/06, number of protein sequences: 4,402). No optional modification was set during the library preparation, but carbamidomethylation was set as a fixed modification and also trypsin/P enzyme with one allowed missed cleavage and a peptide length of 7-30. The false discovery rate (FDR) control was set to 1% FDR. MS1 and MS2 accuracies as well as scan window parameters were set based on the initial test searches (median value from all samples ascertained parameter values). MBR was switched on.

Protein intensities reported in the DIA-NN main report file were further processed using the software container environment (https://github.com/OmicsWorkflows), version 4.7.7a. The processing workflow is available upon request. Briefly, it consisted of: a) the removal of low-quality precursors and contaminant protein groups, b) the normalization of precursor intensities using the loessF algorithm, c) the replacement of missing values with one or more specific values at the precursor level, d) the calculation of protein group MaxLFQ intensities and transformation of these intensities into log2, and e) differential expression analysis using the LIMMA statistical test. Significantly changed protein groups (PGs) had to pass criteria: adjusted p-value (Benjamin-Hochberg correction) < 0.05, log2 FC (fold change) < -1 (downregulation) or > 1 (upregulation). In parallel, data normalization to specific proteins (P0A7V8 and P60438) was also performed. Mass spectrometry proteomics data with the list of all proteins were deposited to the ProteomeXchange Consortium via PRIDE74 partner repository under dataset identifier PXD068732. A source data file containing the list of all proteins is provided with this manuscript.

Isolation of 30S, 50S, and 70S ribosomes for cryo-EM
Ribosomal samples were purified from E. coli MRE600 wt and ΔrimM (rif+kan) strains as previously described75. Briefly, cells were grown in LB medium at 37°C and antibiotics were only added to the overnight cultures to maintain selection: rifampicin (0.5 mg/mL) for both wt and ΔrimM strains, and additionally kanamycin (50 µg/mL) to the ΔrimM strain. The next day, the antibiotic-free LB was inoculated with an overnight culture, and cells were grown to the early exponential phase (OD600 ≈ 0.4). Cells were harvested by centrifugation at 5,000 rpm for 25 minutes at 4 °C (JLA 8.1000 rotor) and washed in ice-cold buffer A (20 mM Tris-HCl, pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 0.5 mM EDTA, 6 mM βME). Afterwards, the cells were lysed and homogenized in a Microfluidics M-110P system in buffer B (20 mM Tris-HCl, pH 7.5, 100 mM NH4Cl, 12.5 mM MgCl2, 0.5 mM EDTA, 6 mM βME). The lysate was clarified by centrifugation at 15,000 rpm for 1 hour at 4°C (JA-17 rotor), followed by filtration through a 0.45 μm membrane to remove residual debris. The clarified lysate was layered onto a 37.7% sucrose cushion prepared in buffer C (20 mM Tris-HCl, pH 7.5, 500 mM NH4Cl, 15 mM MgCl2, 0.5 mM EDTA, 6 mM βME) and ultracentrifuged at 43,000 rpm for 16 h at 4°C (Ti-45 rotor). The ribosomal pellet was washed with buffer B on ice and centrifuged at 4,500 rpm for 5 minutes. After the adjustment of the NH4Cl concentration to 500 mM in 40 ml buffer C, the sample was centrifuged at 55,000 rpm for 2 hours at 4°C (Ti-70 rotor). The resulting pellet was resuspended in 1 ml of buffer B and kept on ice. To isolate the ribosomal samples (30S, 50S and 70S), the resulting ribosomal pellet was layered onto a 10–35% sucrose gradient (in buffer B) and centrifuged at 20,000 rpm for 13 hours at 4°C (SW32-Ti rotor). The fractions of 30S, 50S and 70S were individually collected and concentrated in buffer B (20 mM Tris-HCl, pH 7.5, 100 mM NH4Cl, 12.5 mM MgCl2, 0.5 mM EDTA, 6 mM βME), flash-frozen in liquid nitrogen, and stored at -80°C.

Cryo-EM grid preparation, data collection and image processing
Quantifoil R2/1, 300-mesh grids were glow-discharged for 40 s at ~35 W (30–40 W range) using a Gatan Solarus II. Aliquots of 3.5 μL of 30S, 50S, or 70S samples were applied at a final concentration of 1 μM in buffer A (20 mM Tris-HCl, pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 0.5 mM EDTA, 6 mM βME) on the respective grids. Grids were blotted in a Vitrobot Mark IV (Thermo Fisher Scientific) at 4.5°C and ~95% relative humidity with blot force 0 for 5 seconds, then plunge-frozen in liquid ethane.

All datasets were collected on a Titan Krios (300 kV) equipped with a K3 direct detector and a BioQuantum energy filter (10 eV slit; Gatan). Data were acquired in SerialEM76 at 105,000× nominal magnification (pixel size 0.834 Å) with a defocus range of −0.5 to −2.0 µm. Movies were recorded as 40-frame stacks over 2 s (total dose 40 e⁻/Å²) with continuous streaming. MotionCor277 was used for motion correction and frame averaging. Defocus estimation (CTFFIND4)78 and particle picking were performed in cisTEM (v1.0.0-beta)79. Movies showing ice contamination or excessive drift were excluded based on CTFFIND4 power spectra.

For the 30S datasets, 6,444 micrographs (ΔrimM) and 10,995 micrographs (wt) yielded 767,668 and 1,523,340 particles, respectively. 2D classification into 50 classes in cisTEM retained 455,076 (ΔrimM) and 826,581 (wt) 30S particles. For the 50S datasets, 11,367 (ΔrimM) and 9,710 (wt) micrographs produced 1,523,340 and 1,469,122 particles, with 652,894 (ΔrimM) and 970,120 (wt) 50S particles retained after 2D classification into 50 classes. For the 70S datasets, 15,893 (ΔrimM) and 12,450 (wt) micrographs yielded 2,271,232 and 1,579,782 particles, of which 900,281 (ΔrimM) and 978,750 (wt) 70S particles were retained after 2D classification into 50 classes. Parameter files and particle stacks were then exported from cisTEM, and 4×, 2×, and 1× binned stacks (box size 480 pixels for 1×) were generated using resample.exe from the FREALIGN package (v9.11)80. Particle alignment, 3D classification, and refinement were performed in FREALIGN. Reference maps for 30S, 50S, and 70S were generated in EMAN281 by editing the 70S structure (PDB 6WDE)82 and low-pass filtering to 20 Å. The 4× binned stacks were first aligned to the reference with C1 symmetry over the 300–30 Å range using five global-search (mode 3) cycles, followed by five local-refinement (mode 1) cycles over the 300–18 Å range. The 2× binned stacks were then refined in mode 1 with stepwise high-resolution limits of 18, 12, 10, and 8 Å (five cycles each). The top 60% of particles by alignment score were retained to compute the 3D reconstructions. The final 2× alignment parameters were subsequently used for 3D classification of the 30S, 50S, and 70S particle sets from ΔrimM and wild-type samples.

The 2× binned 30S stacks (ΔrimM and wt) were first classified into 12 classes over 50 cycles (300–8 Å resolution range). For ΔRimM, one high-resolution class containing IF1 and IF3 but lacking uS3, uS10, and uS14 (30SΔrimM - state II) was taken to 1× refinement. Three additional classes were merged (87,412 particles) and underwent an additional round of 3D classification into four subclasses, yielding two high-resolution subclasses. The first subclass contained IF1 and IF3 and was missing uS3, uS10, uS13, uS14, and uS19 (30SΔrimM - state I); and was selected for 1× refinement. The second subclass contained IF1 and IF3 with a complete r-proteins set representing the mature 30S subunit (30SΔrimM - state III); and was selected for 1× refinement.

From the 12 wild-type classes, two were merged (137,111 particles) and subjected to focused mask (IF3 region, 30 Å radius) 3D classification into three subclasses. One high-resolution 30S subclass containing IF1 and IF3 and representing the mature 30S subunit (30Swt) was selected for 1× refinement. Additional focused classifications using spherical or 3D masks placed on the head domain or the mRNA exit tunnel did not yield further classes or improve the overall resolution.

The 2× binned 50S stacks (ΔrimM and wt) were first classified into 16 classes over 50 cycles (300–8 Å resolution range). For ΔrimM, eight classes lacking helix H68 (pre-50SΔrimM; 223,342 particles) were merged and reclassified with a 25 Å focused mask on the RsfS region, yielding three subclasses. One high-resolution subclass with RsfS bound and helix H68 absent (pre-50SΔrimM +RsfS −H68) was taken to 1× refinement. In parallel, five classes with helix H68 present (mature 50SΔrimM; 255,050 particles) were merged and processed identically, producing one high-resolution subclass with RsfS and helix H68 present (50SΔrimM +RsfS +H68) for 1× refinement.

Wild-type 50S data were processed in a similar way. Nine pre-50S classes lacking helix H68 were merged (625,331 particles) and reclassified with a 20 Å focused mask on the RsfS region into three subclasses. One pre-50S subclass containing partial occupancy of RsfS was picked (147,001 particles) and subjected to a second round of focused 3D classification (RsfS region, 20 Å) yielding a high-resolution subclass with RsfS and lacking helix H68 (pre-50Swt +RsfS -H68) for 1× refinement. In the same manner, three classes (152,063 particles) representing mature 50S with helix H68 present were picked and subjected to a focused mask 3D classification (RsfS region, 20 Å radius) and reclassified into three subclasses. One 50Swt subclass with partial occupancy of RsfS and with helix H68 present was picked (255,949 particles) and subjected to a second round of focused mask 3D classification (RsfS region, 20 Å radius). One high-resolution subclass containing RsfS and helix H68 (50Swt +RsfS +H68) was selected for 1× refinement.

The 2× binned 70S stacks (ΔrimM and wt) were first classified into 16 classes over 50 cycles (300–8 Å resolution range). For ΔrimM, three classes were picked, merged (223,385 particles) and reclassified with a 30 Å focused mask encompassing the P and E site tRNA regions into five subclasses. Three high-resolution subclasses were selected and merged into a single class (70SΔrimM) for 1× refinement.

For wild type 70S, 11 classes were picked, merged (582,794 particles) and reclassified with a 20 Å focused mask encompassing the A and P site tRNA regions into four subclasses. One high-resolution class was picked (164,836 particles) and subjected to a second round of 3D classification using a 45 Å focused mask encompassing the P and E site tRNA regions into four subclasses. Three high-resolution subclasses were selected and merged into a single class (70Swt) for 1× refinement. However, no tRNA density was detected in any dataset, likely due to the purification with a high-salt sucrose cushion. Focused 3D classifications with a spherical mask over the tRNA sites yielded no additional classes. Likewise, masking the 30S head domain in 70S ΔrimM and wt stacks did not reveal immature head states or improve overall resolution.

The particles assigned to final high-resolution classes of 30S, 50S and 70S ΔrimM and wt were extracted from the 2x binned stack (with > 50% occupancy and > 0 score) using merge_classes.exe (part of FREALIGN distribution)80. Final refinements of the corresponding unbinned (1×) particle stacks were carried out using mode 1 over 5 cycles. For each, 95% of particles with the highest scores were used to generate the final 3D reconstructions. This process yielded high-resolution cryo-EM maps at ~3.0 Å for 30SΔrimM - state I (12,314 particles); at ~2.9 Å for 30SΔrimM - state II (39,565 particles); at ~2.9 Å for 30SΔrimM - state III (30,144 particles); at ~3.1 Å for 30Swt (43,157 particles); at ~2.7 Å for pre-50SΔrimM +RsfS −H68 (52,567 particles); at ~2.7 Å for 50SΔrimM +RsfS +H68 (51,537 particles); at ~2.7 Å for pre-50Swt +RsfS -H68 (60,038 particles); at ~2.9 Å for 50Swt +RsfS +H68 (11,115 particles); at ~2.6 Å for 70SΔrimM (101,932 particles) and at ~3.0 Å for 70Swt (146,167 particles), according to the 0.143 Fourier Shell Correlation (FSC) criterion, as detailed in Supplementary Tables 3 and 4, and Supplementary Figs. 6, 14. These final cryo-EM maps were used for model building and structure refinements. Local-resolution filtering was applied to the resulting cryo-EM maps, using blocres and blocfilt from the Bsoft (vs. 1.9.1) package83. The sharpening of the resulting cryo-EM maps was performed with bfactor.exe using a B-factor of -30 to -50 Å2 to interpret high-resolution details. FSC curves were calculated with FREALIGN for even and odd particle half-sets.

Model building and refinement
The starting models for refining the 30S, 50S, and 70S ribosomes were derived from the 70S cryo-EM structure (PDB 6WDE)82, which served as the template. The models of IF1, IF3 and RsfS were generated using AlphaFold84 based on E. coli sequences using template structures from PDB 5LMV41 and PDB 6SJ622, respectively. The structural models were initially fitted into the respective cryo-EM maps using rigid-body fitting in Chimera85, while the manual modelling was performed using Coot86. Poorly defined regions within the cryo-EM maps such as rRNA segments, r-proteins or domain regions, were omitted from the models when necessary. All structures were refined using phenix.real_space_refine in Phenix87. Secondary-structure and base-pairing restraints were applied to rRNA, when necessary. Correlation coefficients (model-to-map fit in Phenix) were also manually checked to avoid over-fitting of the models to the cryo-EM maps. FSC values between the final models and cryo-EM maps were computed in Phenix using phenix.mtriage, showing strong agreement between the structural models and cryo-EM maps.

The final models exhibit good stereochemistry, with minimal deviations in bond lengths and angles (Supplementary Tables 3, 4). Model quality was evaluated with MolProbity88. Structural superpositions and figures were prepared in ChimeraX89 and PyMOL (v2.3.1, Schrödinger).

Data availability
The EM density maps generated in this study have been deposited in the EMDB under the following accession codes: EMD-55171 [https://www.ebi.ac.uk/emdb/EMD-55171] (30SΔrimM - State I); EMD-55173 [https://www.ebi.ac.uk/emdb/EMD-55173] (30SΔrimM - State II); EMD-55174 [https://www.ebi.ac.uk/emdb/EMD-55174] (30SΔrimM – State III); EMD-55176 [https://www.ebi.ac.uk/emdb/EMD-55176] (pre-50SΔrimM); EMD-55177 [https://www.ebi.ac.uk/emdb/EMD-55177] (50SΔrimM); EMD-55178 [https://www.ebi.ac.uk/emdb/EMD-55178] (70SΔrimM); EMD-55181 [https://www.ebi.ac.uk/emdb/EMD-55181] (30Swt); EMD-55182 [https://www.ebi.ac.uk/emdb/EMD-55182] (pre-50Swt); EMD-55183 [https://www.ebi.ac.uk/emdb/EMD-55183] (50Swt); and EMD-55185 [https://www.ebi.ac.uk/emdb/EMD-55185] (70Swt). The atomic coordinates generated in this study have been deposited in the PDB under the accession codes 9SS0 [https://doi.org/10.2210/pdb9SS0/pdb] (30SΔrimM - State I); 9SS1 [https://doi.org/10.2210/pdb9SS1/pdb] (30SΔrimM - State II); 9SS2 [https://doi.org/10.2210/pdb9SS2/pdb] (30SΔrimM – State III); 9SS4 [https://doi.org/10.2210/pdb9SS4/pdb] (pre-50SΔrimM); 9SS5 [https://doi.org/10.2210/pdb9SS5/pdb] (50SΔrimM); and 9SS6 [https://doi.org/10.2210/pdb9SS6/pdb] (70SΔrimM). Mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository under dataset identifier PXD068732 [https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD068732]. Source data are also provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors examine the role of RimM, a protein factor previously known to contribute to the assembly of\nthe 30S ribosomal subunit in bacteria, in the restart of growth and in restoring protein synthesis after exposure to adverse\nenvironmental conditions. They discovered that the delta RimM strain demonstrates a significantly longer growth arrest than\nother deletion strains lacking 30S subunit assembly factors (such as RbfA, KsgA, RlmN, RsmE, etc.). Structural analysis of\nthe 30S subunits purified from the delta RimM strain revealed an accumulation of 30S subunits in an immature form, missing\nproteins of the head domain (uS13, uS10, uS19, uS3, and uS14). Interestingly, these particles are bound to IF1 and IF3.\nThe authors propose that these two initiation factors prevent the premature association of these immature 30S subunits with 50S\nsubunits. They also conducted structural analysis of the 50S subunits in the delta RimM strain and compared them to those\nin the wild-type strain. They found that, generally, the 50S subunits in the delta RimM strain are mature, except for a subset\nthat still exhibits H68 in an immature state. A large proportion of 50S subunits in the delta RimM strain are bound to the\nsilencing factor RsfS, and the authors suggest this may be part of the mechanism to prevent premature association of the\n50S subunits with the immature 30S particles.\nOverall, this is a well-conceived study on the role of RimM, an important assembly factor in bacteria. The quality of the\nbiochemical, genetic, and structural analyses conducted by the authors is excellent, but I believe the number of novel\ncontributions in the study does not justify publication in a general-audience journal such as Nature Communications. The\nresearch offers new insights into the molecular mechanisms of RimM's action and elaborates on aspects previously known\nbut lacking detailed analysis or characterization. While these remain valuable contributions to the field of bacterial ribosome\nbiogenesis, they may appeal to a more specialized audience. Below, I review some of these findings and share my\nperspective on their novelty and significance.\n1. The initial part of the results examining the role of RimM in growth recovery is not novel. Publications from the Wikstrom\ngroup from the early 2000s (Bylund et al. (2001) J.Bact. 183:6095-6106) already describe that the delta RimM strain exhibits\na seven-fold reduced growth rate and decreased translation efficiency. This new study confirms Wikstrom’s previous\nfindings and extends them by comparing the growth rates of the delta RimM strain with those of other deletion strains lacking\ndifferent 30S assembly factors.\n2. The cryo-EM analysis of the 30S subunits purified from the delta RimM strain is certainly a welcome expansion of similar\nstudies done in the early 2010s from the Gao (Guo et al. (2013) NAR 41:2609-2620) and Ortega (Leong et al. (2013) RNA\n19:789-802) groups. These earlier cryo-EM studies were done before the recent dramatic technological advances of cryo-\nEM, and the overall resolution of the structures in those studies is limited. The present study characterizes at the molecular\nlevel the structural deficiencies of the immature 30S subunits that accumulate in the delta RimM strain. Therefore, this new\nstudy adds significant granularity to these maturation steps of the head domain assisted by RimM. In my view, the most\nrelevant finding of this study is the finding that these assembly intermediates contain IF1 and IF3 bound to the subunit\ninterface, preventing their association with 50S subunits.\n3. The introduction section that discusses the maturation of the 50S subunit and other assembly factors should be removed\nbecause it is not relevant. This paragraph mentions the role of the ribosome silencing factor RsfS, which binds to uL14 in the\n50S subunit. However, it is already known that RimM does not contribute to 50S subunit maturation. Additionally, the\nobserved increase in RsfS-bound 50S particles in the delta RimM strain is not specific to RimM, as RsfS is known to\ndownregulate translation during stress or stationary phase (Hauser et al., 2012). Therefore, the higher proportion of RsfS-\nbound 50S subunits in the delta RimM strain likely results from cells recovering from slow growth, rather than a direct effect\nor link to RimM's role in maturation. Consequently, the depiction of the 50S subunit bound to RsfS in Fig. 6 as part of the\nrecovery mechanism mediated by RimM is confusing and unsupported by the data.\n4. The sequence of events involving multiple assembly factors contributing to the assembly of the 30S subunit described in\nFig. 1a is highly speculative and unnecessary. The figure legend does not explain the rationale, prior knowledge, or\nreferences considered for this panel. It should be removed.\n5. Suppl. Fig. 10 illustrates the classification strategy of the 30S subunit cryo-EM images obtained from the delta RimM\nstrain. For unexplained reasons, the authors discard many classes that seem to have good resolution and may represent\nclasses of interest. Focusing only on the refinement of the few classes highlighted in yellow could bias the interpretation of\nthis data and, most importantly, ignore relevant biological information regarding the role of RimM contained within those\nclasses. The authors should also consider analysing all these other classes (in grey).\n6. There is an error in the x-axis label of Fig 2e; 0.14 is likely to be 0.1?", "reviewer_2": "In this study, Hassan et al. investigate the mechanism by which deletion of rimM, encoding a ribosome assembly factor,\ncaused delayed recovery of E. coli cells from growth arrest. Using a combination of genetic, biochemical, and structural\napproaches, the authors first show that loss of rimM leads to a pronounced extension of the lag phase and a reduction in\ngrowth rate, more severe than deletions of other ribosome maturation factors. They also observe increased levels of rRNA in\nthe ΔrimM strain. Biochemical assays using an in vitro reconstituted translation system demonstrate that ribosomes\nisolated from ΔrimM cells exhibit impaired translocation during translation elongation. Cryo-EM analysis further reveals that\nboth mature and immature 30S subunits accumulate in the ΔrimM strain. The immature subunits exhibit an incompletely\nformed head domain, consistent with the reduced translocation efficiency observed in vitro. Although previous structural\nstudies of ΔrimM ribosomes existed, they were of limited resolution. This work therefore provides higher-resolution\nstructural models of 30S maturation intermediates and yields mechanistic insights into the stepwise assembly of r-proteins promoted\nby RimM. The authors further show that IF1 and IF3 bind more frequently to 30S subunits isolated from the ΔrimM strain, with\nnotable binding even to immature 30S particles. These observations raise the possibility that initiation factors may\nparticipate in or influence 30S subunit maturation.\nOverall, the manuscript is clearly written and easy to follow. The experiments are carefully controlled, and the combination of\ncomplementary methodologies strengthens the conclusions.\nMajor Comment\nThe authors describe RimM regulates ribosome recovery from growth arrest (e.g., lines 179 and 181). I am not fully\nconvinced that the terms “regulate/regulator” are appropriate here. Efficient ribosome maturation by RimM appears to be a\nhousekeeping function, not a regulatory one. Using “regulate” may suggest a regulatory role analogous to that by ribosome\nhibernation factors, which modulate ribosome activity in response to environmental cues, for example.\nMinor Comments\nLine 129:\nThe notation “E. coli ΔrimM (MG1655)” is ambiguous. It can sound that MG1655 is the name of the ΔrimM strain, although\nthis point becomes clear later in the text.\nIf there is a specific reason why ribosome purification was performed using MRE600 rather than an MG1655 derivative, it\nmay be helpful to briefly explain the rationale.", "reviewer_3": "I am not an expert in structural biology, therefore I will not comment on the Cryo-EM part of the manuscript. I focus my review\non the structure supporting experiments.\nIn the manuscript entitled ‘Mechanistic insights into E. coli recovery from growth arrest’ by Hassan et al. authors investigate\nthe role of RimM ribosome biogenesis factor using Cryo-EM and in vivo and in vitro experiments.\nThe authors conclude that cells lacking ribosomes processed by RimM protein take a longer time, compared to the wild-type\ncells, to enter the logarithmic growth phase. They present a prolonged lag phase. Experiments presented in the manuscript\nare performed correctly.\nHowever, I have some questions about the conclusions.\n1. Authors discuss recovery from growth arrest but they do not spend any time investigating what actually happened in the\nstationary phase to the ribosomes from delta-rimM cells. This is crucial and this is where they should look into in this\nmanuscript.\n2. Cells in a delta-rimM strain take longer to restart growth. What happens to the ribosomes in the stationary phase? Is rRNA\ndegraded, truncated? Do ribosomes have problems hibernating to sustain their function? Authors should check the stability\nof rRNA in the stationary phase, for example using capillary gel electrophoresis (like for example, Bioanalyser). Is rRNA\nmore prone for degradation in the stationary phase? Based on the structure, can you predict rRNA degradation (if\nconfirmed)?\n3. Do ribosomes in delta-rimM form 100S hibernation complexes (compare to the wt)? Based on the structure, can you\npredict if the binding of the hibernation factors will be disturbed? Are hibernation factors downregulated in MS? (I am aware\nthe data is not from the stationary phase).\n4. Sucrose gradient profiles from the stationary phase, and OD 0.1 and 1 are missing. Stationary phase data and OD 0.1\nwould be crucial to determine what happened to the ribosomes in the stationary phase.\n5. Mass spec data is not available; authors should provide a dataset in the submission set (for example Excel dataset).\n6. Authors use OD600 to normalize cell density for comparative experiments (for example, RNA, protein content per cell).\nHave the authors checked the relation of OD and colony-forming units? Cells with different size scatter light differently."}, "author_response": "Point-by-point response to the reviewer comments\nReviewer #1 (Remarks to the Author):\nOverall, this is a well-conceived study on the role of RimM, an important assembly\nfactor in bacteria. The quality of the biochemical, genetic, and structural analyses\nconducted by the authors is excellent, but I believe the number of novel contributions\nin the study does not justify publication in a general-audience journal such as Nature\nCommunications. The research offers new insights into the molecular mechanisms of\nRimM's action and elaborates on aspects previously known but lacking detailed\nanalysis or characterization. While these remain valuable contributions to the field of\nbacterial ribosome biogenesis, they may appeal to a more specialized audience.\nBelow, I review some of these findings and share my perspective on their novelty and\nsignificance.\n1. The initial part of the results examining the role of RimM in growth recovery is not\nnovel. Publications from the Wikstrom group from the early 2000s (Bylund et al. (2001)\nJ.Bact. 183:6095-6106) already describe that the delta RimM strain exhibits a seven-\nfold reduced growth rate and decreased translation efficiency. This new study confirms\nWikstrom’s previous findings and extends them by comparing the growth rates of the\ndelta RimM strain with those of other deletion strains lacking different 30S assembly\nfactors.\nWe appreciate the reviewer’s comments regarding the growth recovery phenotype of\nbacterial strain lacking the 30S biogenesis factor RimM. While we acknowledge that a\nslow-growth phenotype of ΔrimM strain has been reported previously, we believe the\nnovelty of our study lies in the systematic comparison of this phenotype with strains\nlacking other 30S biogenesis factors and/or ribosomal proteins. This direct comparison\nreveals that loss of RimM produces the most severe recovery defect among the factors\nexamined, underscoring the critical importance of proper maturation and folding of the\n3′ head domain of the 30S subunit for efficient translation. Notably, deletion of the\nprimary head-domain ribosomal protein uS9 or of RbfA—often considered a central\nplayer in 30S biogenesis—does not result in a comparable phenotype. In addition, by\nanalyzing ribosomes isolated from lag, early exponential, and late exponential growth\nphases, we provide mechanistic insight into why defects in the 30S head domain must\nbe resolved to restore efficient translocation of tRNAs and mRNA during recovery. We\nbelieve that these combined functional insights offer a comprehensive perspective on\nthe role of RimM in 30S biogenesis and are well suited for a broad readership, such as\nthat of Nature Communications.\n2. The cryo-EM analysis of the 30S subunits purified from the delta RimM strain is\ncertainly a welcome expansion of similar studies done in the early 2010s from the\nGao (Guo et al. (2013) NAR 41:2609-2620) and Ortega (Leong et al. (2013) RNA 19:789-\n802) groups. These earlier cryo-EM studies were done before the recent dramatic\ntechnological advances of cryo-EM, and the overall resolution of the structures in those\nstudies is limited. The present study characterizes at the molecular level the structural\ndeficiencies of the immature 30S subunits that accumulate in the delta RimM strain.\nTherefore, this new study adds significant granularity to these maturation steps of the\nhead domain assisted by RimM. In my view, the most relevant finding of this study is\nthe finding that these assembly intermediates contain IF1 and IF3 bound to the subunit\ninterface, preventing their association with 50S subunits.\nWe thank the reviewer for this assessment and for placing our work in the context of\nthe pioneering studies by the Gao and Ortega groups. We fully agree that these earlier\ninvestigations provided important initial insights into 30S assembly intermediates in the\nabsence of RimM; however, they were necessarily limited by both resolution and\nscope, and focused exclusively on characterizing 30S maturation defects. To our\nknowledge, no previous study has attempted to comprehensively address how cells\nultimately assemble functional 70S ribosomes and restore translation under conditions\nof perturbed 30S biogenesis.\nThe central advance of our study lies precisely in this broader perspective. By\nresolving, at near-atomic resolution, a complete and internally consistent set of\nribosomal particles—30S, 50S, and 70S—isolated from the ΔrimM background, we\nmove to directly interrogate how ribosome assembly, quality control, and translational\nrecovery are coordinated. Importantly, parallel analysis of ribosomal particles isolated\nfrom wild-type cells provides a critical reference framework that allows us to distinguish\ndefective and fully functional states, and to directly assess how productive 70S\nribosomes are assembled despite impaired 30S maturation.\nWithin this framework, our cryo-EM analysis resolves discrete maturation states of the\n30S subunit that capture progressive folding of the 3′ major domain of the 16S rRNA\nand stepwise incorporation of late-binding head-domain ribosomal proteins. While\nsome structural features are consistent with earlier reports, the improved resolution\nenables localization of rRNA disorder, protein occupancy, and factor binding, allowing\nthese intermediates to be placed into a functional context that was previously\ninaccessible.\nImportantly, and as highlighted by the reviewer, we consider the most significant finding\nof this work to be the association of IF1 and IF3 with immature 30S particles. Our\nstructures show that these initiation factors remain bound at the subunit interface\nacross multiple maturation states, thereby preventing premature association with the\n50S subunit. This provides a direct structural mechanism explaining how cells avoid\nthe formation of defective 70S ribosomes while still permitting recovery of translational\ncapacity once properly assembled subunits become available.\nCrucially, this anti-association checkpoint was not resolved, despite similar purification\nstrategies, and likely reflects both the improved resolving power of current cryo-EM\nmethodologies and our deliberate focus on ribosomal particles isolated during early\nexponential recovery. By integrating high-resolution structural analysis with functional\nassays, quantitative proteomics, and growth-phase–resolved ribosome profiling, our\nstudy offers a comprehensive view of how bacteria preserve translational fidelity and\nrestore protein synthesis when 30S biogenesis is perturbed. We believe that this\nintegrated conceptual and methodological framework provides a broadly applicable\nstrategy for investigating translation recovery and ribosome quality control under stress\nconditions and will be of interest to a broad readership.\n3. The introduction section that discusses the maturation of the 50S subunit and other\nassembly factors should be removed because it is not relevant. This paragraph\nmentions the role of the ribosome silencing factor RsfS, which binds to uL14 in the\n50S subunit. However, it is already known that RimM does not contribute to 50S subunit\nmaturation. Additionally, the observed increase in RsfS-bound 50S particles in the\ndelta RimM strain is not specific to RimM, as RsfS is known to downregulate translation\nduring stress or stationary phase (Hauser et al., 2012). Therefore, the higher proportion\nof RsfS-bound 50S subunits in the delta RimM strain likely results from cells recovering\nfrom slow growth, rather than a direct effect or link to RimM's role in maturation.\nConsequently, the depiction of the 50S subunit bound to RsfS in Fig. 6 as part of the\nrecovery mechanism mediated by RimM is confusing and unsupported by the data.\nWe thank the reviewer for this important comment and agree that RimM is not directly\ninvolved in 50S subunit maturation. In response, we have removed the introductory\ntext describing 50S biogenesis and late-stage 50S assembly factors, as it was not\nessential for the central focus of the study.\nWe have revised the remaining text to clarify that our interest in the 50S subunit does\nnot concern its maturation, but rather whether perturbation of 30S biogenesis elicits a\ncoordinated cellular response that limits premature subunit joining. Accordingly, the\ndiscussion of RsfS has been reformulated to emphasize its established role as a\nstress-responsive anti-association factor, rather than implying a RimM-specific function\nin 50S assembly.\nConsistent with prior work (including Hauser et al., 2012), we now explicitly state that\nthe increased occupancy of RsfS observed in the ΔrimM strain reflects a general\nadaptive response to slow growth, rather than a direct mechanistic link between RimM\nand RsfS. In this context, deletion of RimM induces prolonged recovery and impaired\n30S maturation, which in turn triggers upregulation of anti-association mechanisms—\nincluding RsfS—to prevent premature formation of defective 70S ribosomes.\nWe have also revised Figure 6 and its legend to avoid suggesting that RsfS acts as\npart of a RimM-specific recovery pathway. Instead, the figure now illustrates how RsfS\nfunctions alongside IF1 and IF3 as part of a broader, stress-induced safeguarding\nprogram that restrains 70S assembly when 30S biogenesis is compromised.\nTogether, these revisions clarify that our findings do not attribute a novel role of RimM\nin 50S maturation, but rather demonstrate how impaired 30S assembly engages\nconserved anti-association pathways to ensure that only properly matured ribosomal\nsubunits enter translation.\n4. The sequence of events involving multiple assembly factors contributing to the\nassembly of the 30S subunit described in Fig. 1a is highly speculative and\nunnecessary. The figure legend does not explain the rationale, prior knowledge, or\nreferences considered for this panel. It should be removed.\nIn response to the reviewer’s comment, Figure 1a has been removed to avoid\nspeculative or potentially misleading interpretation of the presented data.\n5. Suppl. Fig. 10 illustrates the classification strategy of the 30S subunit cryo-EM\nimages obtained from the delta RimM strain. For unexplained reasons, the authors\ndiscard many classes that seem to have good resolution and may represent classes\nof interest. Focusing only on the refinement of the few classes highlighted in yellow\ncould bias the interpretation of this data and, most importantly, ignore relevant\nbiological information regarding the role of RimM contained within those classes.\nThe authors should also consider analysing all these other classes (in grey).\nWe understand the concern raised by the reviewer and would like to clarify how these\nclasses were handled. We carefully and systematically analyzed all additional particle\nclasses shown in grey to avoid bias or omission in our interpretation of 30S maturation\nin the absence of RimM. Many of these classes were merged into smaller stacks and\nsubjected to multiple rounds of focused 3D classification, using masks targeting either\nthe 30S head domain or the mRNA tunnel, in an effort to identify any additional\nstructurally or biologically relevant states. However, these classes consistently\nrecapitulated features of 30S maturation intermediates that have already been\ndescribed in earlier studies, including those reported by Guo et al., NAR, (2013) and\nLeong et al., RNA, (2013), as noted in the Results section. We therefore focused our\nanalysis on additional classes that are particularly relevant to the control of 70S\nassembly, as these states not only reflect sequential ribosomal protein incorporation\nbut also reveal the involvement of initiation factors acting as anti-association elements.\nNotably, such states were not resolved in previous studies, despite similar purification\nstrategies. By collecting 30S samples at OD = 0.4, our dataset captures high-\n600\nresolution structural states that were previously omitted and that provide new insight\ninto a mechanism that safeguards 70S assembly.\n6. There is an error in the x-axis label of Fig 2e; 0.14 is likely to be 0.1?\nWe thank the reviewer for noting this typo error; it has been corrected in panel e of\nFigure 2.\nReviewer #2 (Remarks to the Author):\nIn this study, Hassan et al. investigate the mechanism by which deletion of rimM,\nencoding a ribosome assembly factor, caused delayed recovery of E. coli cells from\ngrowth arrest. Using a combination of genetic, biochemical, and structural approaches,\nthe authors first show that loss of rimM leads to a pronounced extension of the lag\nphase and a reduction in growth rate, more severe than deletions of other ribosome\nmaturation factors. They also observe increased levels of rRNA in the ΔrimM strain.\nBiochemical assays using an in vitro reconstituted translation system demonstrate that\nribosomes isolated from ΔrimM cells exhibit impaired translocation during translation\nelongation. Cryo-EM analysis further reveals that both mature and immature 30S\nsubunits accumulate in the ΔrimM strain. The immature subunits exhibit an\nincompletely formed head domain, consistent with the reduced translocation efficiency\nobserved in vitro. Although previous structural studies of ΔrimM ribosomes existed,\nthey were of limited resolution. This work therefore provides higher-resolution\nstructural models of 30S maturation intermediates and yields mechanistic insights into\nthe stepwise assembly of r-proteins promoted by RimM. The authors further show that\nIF1 and IF3 bind more frequently to 30S subunits isolated from the ΔrimM strain, with\nnotable binding even to immature 30S particles. These observations raise the\npossibility that initiation factors may participate in or influence 30S subunit maturation.\nOverall, the manuscript is clearly written and easy to follow. The experiments are\ncarefully controlled, and the combination of complementary methodologies\nstrengthens the conclusions.\nMajor Comment\nThe authors describe RimM regulates ribosome recovery from growth arrest (e.g., lines\n179 and 181). I am not fully convinced that the terms “regulate/regulator” are\nappropriate here. Efficient ribosome maturation by RimM appears to be a\nhousekeeping function, not a regulatory one. Using “regulate” may suggest a\nregulatory role analogous to that by ribosome hibernation factors, which modulate\nribosome activity in response to environmental cues, for example.\nWe thank the reviewer for this important note. We agree that the terms “regulate” and\n“regulator” may imply a conditional or stress-responsive role that is not appropriate for\nRimM. RimM functions as a constitutive 30S biogenesis factor required for efficient\nribosome maturation, rather than as a regulatory factor that modulates ribosome\nactivity in response to environmental cues, such as ribosome hibernation factors.\nAccordingly, we have revised the manuscript to remove or replace “regulate/regulator”\nwhere it referred to RimM. These instances have been rephrased to describe RimM as\nbeing “required for,” or “supporting” efficient ribosome maturation and recovery from\ngrowth arrest. This revised wording more accurately reflects RimM’s housekeeping\nrole in 30S assembly and avoids conflating its function with stress-responsive\nregulatory mechanisms.\nMinor Comments\nLine 129:\nThe notation “E. coli ΔrimM (MG1655)” is ambiguous. It can sound that MG1655 is the\nname of the ΔrimM strain, although this point becomes clear later in the text.\nWe thank the reviewer for pointing this out. We have revised the wording to clearly\nstate that the ΔrimM was constructed in the E. coli MG1655 background, thereby\navoiding any confusion that MG1655 might refer to the mutant strain name itself.\nIf there is a specific reason why ribosome purification was performed using MRE600\nrather than an MG1655 derivative, it may be helpful to briefly explain the rationale.\nWe have added a brief explanation in the Results section describing the structural\nanalysis of 30S intermediates, clarifying that ribosome purification was performed\nusing the E. coli MRE600 background because of its reduced ribonuclease activity,\nwhich may facilitate preservation of 30S assembly intermediates and is also relevant\nfor subsequent purification and functional validation of 70S ribosomes in kinetic\nassays.\nReviewer #3 (Remarks to the Author):\nI am not an expert in structural biology, therefore I will not comment on the Cryo-EM\npart of the manuscript. I focus my review on the structure supporting experiments.\nIn the manuscript entitled ‘Mechanistic insights into E. coli recovery from growth arrest’\nby Hassan et al. authors investigate the role of RimM ribosome biogenesis factor using\nCryo-EM and in vivo and in vitro experiments.\nThe authors conclude that cells lacking ribosomes processed by RimM protein take a\nlonger time, compared to the wild-type cells, to enter the logarithmic growth phase.\nThey present a prolonged lag phase. Experiments presented in the manuscript are\nperformed correctly.\nHowever, I have some questions about the conclusions.\n1. Authors discuss recovery from growth arrest but they do not spend any time\ninvestigating what actually happened in the stationary phase to the ribosomes from\ndelta-rimM cells. This is crucial and this is where they should look into in this\nmanuscript.\nWe thank the reviewer for raising this important point and fully agree that\nunderstanding ribosome behavior during stationary phase in the ΔrimM background is\nbiologically highly relevant. The primary goal of the present study, however, is to define\nhow cells restore functional translation and productive 70S ribosome assembly upon\nexit from growth arrest when 30S biogenesis is perturbed by loss of RimM.\nAccordingly, our study focuses on the recovery phase, during which ribosomes\ntransition from inactive or immature states to translationally competent 70S complexes.\nThis window enabled us to identify a structural and functional checkpoint characterized\nby delayed 30S head-domain maturation and sustained binding of anti-association\nfactors (IF1, IF3, and RsfS), which together prevent premature or defective 70S\nassembly. We therefore deliberately concentrated on this phase to comprehensively\ndemonstrate, using complementary structural, biochemical, and kinetic approaches,\nhow the translation machinery is restored under conditions of impaired 30S biogenesis.\nHowever, as pointed out by the reviewer, the ribosome remodeling during stationary\nphase represents a distinct and biologically important question. We have already\nbegun investigating this growth phase in the ΔrimM strain; however, our intention is to\naddress it as a separate study in which we can comprehensively examine how\nribosome hibernation is altered or reprogrammed under conditions of impaired 30S\nbiogenesis. Such an analysis will require an integrated structural and biochemical\napproach to define the mechanisms that govern translational silencing and\npreservation during stationary phase.\nIn the present manuscript, which focuses on translation reactivation during the\nrecovery phase, we therefore limit our analysis of stationary phase to polysome\nprofiling across multiple growth stages (Supplementary Fig. 4). These data reveal that\nthe hibernation scheme is markedly altered in ΔrimM cells, highlighting the need for\nfurther detailed investigation into the factors and mechanisms that control this\nreprogramming, which is currently ongoing in our lab.\n2. Cells in a delta-rimM strain take longer to restart growth. What happens to the\nribosomes in the stationary phase? Is rRNA degraded, truncated? Do ribosomes have\nproblems hibernating to sustain their function? Authors should check the stability of\nrRNA in the stationary phase, for example using capillary gel electrophoresis (like for\nexample, Bioanalyser). Is rRNA more prone for degradation in the stationary phase?\nBased on the structure, can you predict rRNA degradation (if confirmed)?\nWe fully agree with the reviewer that assessing rRNA stability and degradation during\nstationary phase in the ΔrimM strain is an important direction. In the current study, we\ndid not perform direct measurements of rRNA integrity in stationary phase as our\nprimary focus was on the recovery phase and the structural mechanisms enabling\nreactivation of translation.\nThat said, our stationary phase polysome profiles (Supplementary Fig. 4) reveal\nreduced formation of hibernating 100S complexes and an altered balance of free 30S\nand 50S subunits in ΔrimM cells, suggesting that ribosome preservation during\nstationary phase may indeed be compromised. In the Discussion, we cautiously\npostulate that this phenotype could be linked to a pool of incompletely matured 30S\nsubunits—particularly those with defects in the 3′ end of the 16S rRNA—which may be\nmore susceptible to ribonuclease-mediated turnover.\nThis hypothesis is further supported by our proteomic data (Supplementary Fig. 15)\nshowing downregulation of canonical hibernation factors (HPF, RaiA) during early\nexponential growth, potentially predisposing ribosomes entering stationary phase to\nreduced protection. However, we fully agree that direct analysis of rRNA stability and\ndegradation in stationary phase is required to validate this model. Such experiments,\nincluding biochemical assays of rRNA integrity, are a major focus of our ongoing work\nand are intentionally separated from the recovery-focused narrative of the present\nmanuscript.\n3. Do ribosomes in delta-rimM form 100S hibernation complexes (compare to the wt)?\nBased on the structure, can you predict if the binding of the hibernation factors will be\ndisturbed? Are hibernation factors downregulated in MS? (I am aware the data is not\nfrom the stationary phase).\nWe thank the reviewer for raising this important question. Polysome profiling\ndemonstrates that ΔrimM cells are capable of forming 100S ribosome hibernation\ncomplexes; however, their abundance is reduced in stationary phase compared to\nwild-type cells, accompanied by an altered distribution of free 30S and 50S subunits\n(Supplementary Fig. 4). These observations are now presented and discussed in the\nrevised manuscript.\nConsistent with these findings, our mass spectrometry analysis performed during early\nexponential growth reveals downregulation of several canonical ribosome hibernation\nfactors in the ΔrimM background. Although these measurements were not conducted\nin stationary phase, they suggest that ribosomes entering stationary phase in ΔrimM\ncells may already be insufficiently protected, potentially compromising the efficiency or\nstability of hibernation. For clarity, we have updated the Supplementary Figure 15 to\nhighlight hibernation and ribosome quality control associated factors, including Hpf,\nRaiA, RNase R, and YebY.\nBased on our structural data, we do not predict direct disruption of hibernation factor\nbinding sites on mature ribosomes. Instead, as discussed in the manuscript, we\npropose that incomplete 30S maturation combined with altered factor availability may\nindirectly affect ribosome hibernation, in line with previous observations (Prossliner et\nal., NAR, 2021). Importantly, we note that the effects of rimM deletion are not limited\nto hibernation alone, but are also likely to influence ribosome collisions and associated\nquality control pathways.\nWe emphasize that these interpretations are made cautiously. A comprehensive\nunderstanding of how disrupted 30S biogenesis impacts ribosome hibernation and\nquality control will require extensive structural analyses as well as complementary\nbiochemical and rRNA degradation studies. As noted, such investigations are currently\nunderway in our laboratories to generate a full comprehensive story.\n4. Sucrose gradient profiles from the stationary phase, and OD 0.1 and 1 are missing.\nStationary phase data and OD 0.1 would be crucial to determine what happened to the\nribosomes in the stationary phase.\nWe agree with the reviewer that growth-phase resolved ribosome profiles are\nimportant. We have therefore included polysome profiling data from ΔrimM cells\nharvested at OD = 0.1 (termed lag/recovery phase), OD = 1.0 (termed late\n600 600\nexponential phase), and stationary phase (Supplementary Fig. 4). These data\ndemonstrate that ribosome distributions at OD = 0.1 and 1.0 closely resemble those\n600\nobserved at OD = 0.4, validating that the structural analyses performed at OD =\n600 600\n0.4 are representative. In contrast, stationary phase profiles show a distinct\nredistribution of ribosomal species, particularly affecting 100S formation and the\nbalance of free subunits. We now explicitly present and discuss these data prior to the\nstructural description of 30S intermediates, which we identify as an important avenue\nfor future investigation.\n5. Mass spec data is not available; authors should provide a dataset in the submission\nset (for example Excel dataset).\nWe have now included the complete processed mass spectrometry datasets as Excel\ntable and deposited it as a raw source MS file for access directly linked to this\nmanuscript.\n6. Authors use OD600 to normalize cell density for comparative experiments (for\nexample, RNA, protein content per cell). Have the authors checked the relation of OD\nand colony-forming units? Cells with different size scatter light differently.\nWe thank the reviewer for raising this important point regarding the use of OD as a\n600\nproxy for cell number. First, to address the concern that differences in cell size between\nwt and ΔrimM strains might affect the relationship between OD and cell number, we\n600\ndirectly compared colony-forming units (CFUs) at matched OD values. As shown in\n600\nduring growth at 37°C, diluted, and plated on LB agar. At the same dilution (10⁻⁴), both\nstrains yielded comparable numbers of colonies, indicating similar CFU per unit OD .\n600\nof viable cells in wt and ΔrimM liquid cultures. Differences in colony size and plate\nincubation time reflect differences in growth rate on solid medium and do not affect the\nCFU-based estimation of cell number in liquid culture. Second, with regard to the use\nof OD for normalization, we would like to clarify that OD was not used to normalize\n600 600\ncellular RNA or protein content across samples collected at different optical densities.\nInstead, OD was used solely to monitor growth and to harvest cells at defined growth\n600\nphases. All comparative analyses of RNA and protein content were performed on\nsamples collected at comparable growth stages, thereby avoiding normalization across\ndifferent OD conditions. Additionally, a clarification sentence was added in the viability\nassay method section."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I appreciate the revisions these authors made in response to my feedback and comments. In the revised version and the\npoint-by-point response letter, it is now clear how this new study significantly expands earlier work from the Wikstrom lab on\nthe slow-growth phenotype of the delta RimM strain. Based on their responses, I agree that this paper represents a\nsignificant expansion of this particular aspect, including a systematic comparison of this phenotype with strains lacking other\n30S biogenesis factors and/or ribosomal proteins. None of this has been reported before. Similarly, cryo-EM analysis of the\n30S subunits purified from the delta RimM strain significantly expands our structural understanding of these ribosomal\nparticles compared with lower-resolution studies around 2013 from the Guo and Ortega groups. I also welcome the changes\nthe authors have made regarding their interpretation of the 50S subunits purified from the delta RimM strain and their\nassociation with RsfS. I think the text describing these findings is now well adjusted and consistent with the existing\nliterature on RsfS.\nOverall, all my concerns are addressed, and I am supportive of the publication of this revised version of the manuscript as is.", "reviewer_2": "The authors have addressed all my comments.", "reviewer_3": "The reply to my comments by authors of the the manuscript entitled ‘Mechanistic insights into E. coli recovery from growth\narrest’ by Hassan et al. is satisfactory.\nThis is a complex, comprehensive work which should be published.\nMinor comment:\nline 312 RsfS controls the 50S subunit association and 70S assembly\nAssociation with? it should be written down.", "reviewer_4": "LC-MS/MS Technical Review: Mechanistic insights into E. coli recovery from growth arrest\nHassan et. al use LC-MS/MS to probe the differential expression between wild type E. coli and E. coli ΔrimM strain, which\nexhibits a prolonged growth arrest. Their work complements other broader experimentation. The researchers use a modern\nBruker Tims TOF HT mass spectrometer with a nanoLC and long gradient and a DIA scanning method. The hardware is up\nto date, and the DIA parameters are standard and easily reproduced on such an instrument. Three biological replicates are\nused, and the sample prep follows established protocols. The data is also mined using standard search methods and\nfollows a logical progression of protocols and statistics to arrive at the result of protein expression differences. As such, the\noverall LC-MS/MS technical methods are current and accurate.\nMinor issues with the technical experimentation are as follows:\n(1) The supplemental information in PRIDE contains 12 raw mass spectrometry files and the sample information supports\nthat. However, the experimental section is based on 2 strains in triplicate, so the question is why are the additional raw files\nprovided?\n(2) Authors use iRT + trypsin database presumably for retention correction/normalization. However, it is not written in the\nsample prep if these iRT peptides are spiked in or not. As such, clarification is warranted.\n(3) In the statistical write up section of the LC-MS/MS methods the authors should include: what is the method used for\nmultiple testing correction (eg. Benjamini-Hochberg, other), what is the cut off for significant protein FDR and fold change\nand which category of proteins were filtered in or out and annotated in the volcano plot. To this effect, a list of all proteins\nfound should be provided in PRIDE.\n(4) The coloring scheme of the Volcano plot is very light and hard to read. A more contrasting color palette would make it\neasier for readers."}, "author_response": "Point-by-point response to the reviewer comments\nReviewer #1 (Remarks to the Author):\nI appreciate the revisions these authors made in response to my feedback and\ncomments. In the revised version and the point-by-point response letter, it is now clear\nhow this new study significantly expands earlier work from the Wikstrom lab on the\nslow-growth phenotype of the delta RimM strain. Based on their responses, I agree\nthat this paper represents a significant expansion of this particular aspect, including a\nsystematic comparison of this phenotype with strains lacking other 30S biogenesis\nfactors and/or ribosomal proteins. None of this has been reported before. Similarly,\ncryo-EM analysis of the 30S subunits purified from the delta RimM strain significantly\nexpands our structural understanding of these ribosomal particles compared with\nlower-resolution studies around 2013 from the Guo and Ortega groups. I also welcome\nthe changes the authors have made regarding their interpretation of the 50S subunits\npurified from the delta RimM strain and their association with RsfS. I think the text\ndescribing these findings is now well adjusted and consistent with the existing literature\non RsfS.\nOverall, all my concerns are addressed, and I am supportive of the publication of this\nrevised version of the manuscript as is.\nWe thank the reviewer for the constructive remarks throughout the review process and\nappreciate the opportunity to address all of the concerns raised.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed all my comments.\nWe thank the reviewer for the constructive remarks throughout the review process and\nappreciate the opportunity to address all of the concerns raised.\nReviewer #3 (Remarks to the Author):\nThe reply to my comments by authors of the the manuscript entitled ‘Mechanistic\ninsights into E. coli recovery from growth arrest’ by Hassan et al. is satisfactory. This\nis a complex, comprehensive work which should be published.\nMinor comment:\nline 312 RsfS controls the 50S subunit association and 70S assembly\nAssociation with? it should be written down.\nWe thank the reviewer for the constructive remarks throughout the review process.\nThe subheading has been revised to: “RsfS controls the association of the 50S subunit\nwith the 30S subunit and the 70S assembly.”\nReviewer #4 (Remarks to the Author):\nLC-MS/MS Technical Review: Mechanistic insights into E. coli recovery from growth\narrest\nHassan et. al use LC-MS/MS to probe the differential expression between wild type E.\ncoli and E. coli ΔrimM strain, which exhibits a prolonged growth arrest. Their work\ncomplements other broader experimentation. The researchers use a modern Bruker\nTims TOF HT mass spectrometer with a nanoLC and long gradient and a DIA scanning\nmethod. The hardware is up to date, and the DIA parameters are standard and easily\nreproduced on such an instrument. Three biological replicates are used, and the\nsample prep follows established protocols. The data is also mined using standard\nsearch methods and follows a logical progression of protocols and statistics to arrive\nat the result of protein expression differences. As such, the overall LC-MS/MS technical\nmethods are current and accurate.\nMinor issues with the technical experimentation are as follows:\n(1) The supplemental information in PRIDE contains 12 raw mass spectrometry files\nand the sample information supports that. However, the experimental section is based\non 2 strains in triplicate, so the question is why are the additional raw files provided?\nWe thank the reviewer for raising this point and apologize for the lack of clarity in the\noriginal description. In this study, two E. coli strains - Escherichia coli K-12 MG1655\n(MG1655) and Escherichia coli MRE600 (MRE600)- were analyzed in biological\ntriplicates. For each strain, two variants were examined (wt and ΔrimM), resulting in a\ntotal of 12 LC–MS/MS measurements (2 strains × 2 variants × 3 biological replicates).\nAccordingly, 12 raw files were generated and deposited in PRIDE. We have now\nrevised the Methods section to explicitly describe this experimental design. In the\nmanuscript, we present only the volcano plot derived from the MRE600 dataset (wt vs.\nΔrimM). This is because the cryo-EM experiments of ribosomal particles were\nperformed using the MRE600 strain, and we therefore focused on this dataset to\nensure a direct correlation between the structural and proteomic analyses. The\nMG1655 dataset was nevertheless deposited in PRIDE to support transparency and\nopen science. Although it shows comparable trends and is not directly linked to the\nstructural work presented here, we consider it valuable to make these raw data publicly\navailable for the scientific community.\n(2) Authors use iRT + trypsin database presumably for retention\ncorrection/normalization. However, it is not written in the sample prep if these iRT\npeptides are spiked in or not. As such, clarification is warranted.\nWe thank the reviewer for pointing out this omission. We agree that the use of iRT\npeptides was not described with sufficient clarity in the original sample preparation\nsection. The Methods section has now been revised to explicitly state that iRT peptides\nwere spiked into each sample prior to LC–MS/MS analysis.\n(3) In the statistical write up section of the LC-MS/MS methods the authors should\ninclude: what is the method used for multiple testing correction (eg. Benjamini-\nHochberg, other), what is the cut off for significant protein FDR and fold change\nand which category of proteins were filtered in or out and annotated in the volcano plot. To\nthis effect, a list of all proteins found should be provided in PRIDE.\nThe Methods section has been revised to explicitly describe the statistical procedures\nused for the LC–MS/MS analysis. Specifically, we now state the method used for\nmultiple testing correction, the false discovery rate (FDR) cutoff applied for\nsignificance, and the fold-change threshold used to define differential protein\nabundance. Regarding the volcano plot, we apologize for the previously unclear\nlabeling. The grey open circles do not represent filtered-out proteins; rather, they\ncorrespond to all other proteins detected by the MS analysis that are not specifically\nhighlighted in the figure. These proteins were not individually annotated because they\nare not directly relevant to the main focus of the manuscript. We have now clarified this\nin the legend of Supplementary Figure 15, where it is explicitly stated that grey circles\nrepresent all other detected proteins, while only proteins relevant to this study are\nhighlighted in colored dots and labeled. Finally, in line with the reviewer’s request and\nto ensure full data transparency, the complete list of all detected proteins has been\ndeposited in the PRIDE repository and is also provided as a source data file\naccompanying the manuscript.\n(4) The coloring scheme of the Volcano plot is very light and hard to read. A more\ncontrasting color palette would make it easier for readers.\nWe thank the reviewer for this helpful suggestion. To improve the clarity and readability\nof the volcano plot, we have revised the color scheme in Supplementary Figure 15.\nSpecifically, we increased the contrast of the color palette by using darker colors for\nthe data points and open circles, and we relabelled the key proteins using labels with\na black background. These changes enhance the visibility of the highlighted proteins\nand make the figure easier to interpret, particularly for the proteins that are most\nrelevant to the conclusions of this manuscript."}]

Decision letter