NSUN4 Is a Dual Function Mitochondrial Protein Required for Both Methylation of 12S rRNA and Coordination of Mitoribosomal Assembly

Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m⁵C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.     

MPV17 Loss Causes Deoxynucleotide Insufficiency and Slow DNA Replication in Mitochondria

MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17^-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17^-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients’ quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway.     

Mitochondrial ribosome assembly in Neurospora crassa. Chloramphenicol inhibits the maturation of small ribosomal subunits.

Recent results suggest that, in Neurospora crassa, one small subunit mitochondrial ribosomal protein (S-4a, M_r 52,000) is synthesized intramitochondrially (Lambowitz et al., 1976). We now find that, when wild-type cells are treated with chloramphenicol to block mitochondrial protein synthesis, the maturation of 30 S mitochondrial ribosomal subunits is rapidly inhibited and there is an accumulation of CAP-30 S particles which sediment slightly behind mature small subunits. Electrophoretic analysis suggests that the CAP-30 S particles are deficient in several proteins including S-4a and that they are enriched in a precursor RNA species that is slightly longer than 19 S RNA. Chloramphenicol also appears to inhibit the maturation of 50 S ribosomal subunits, but this effect is much less pronounced. Continued incubation in chloramphenicol leads to a decrease in the proportion of total mitochondrial ribonucleoprotein present as monomers, possibly reflecting the depletion of competent subunits. After long-term (17 h) growth in chloramphenicol, mitochondrial ribosome profiles from wild-type cells show decreased ratios of small to large subunits, a feature which is also characteristic of the poky (mi-1) mutant. Pulse-labeling experiments combined with electrophoretic analysis show that the synthesis of mitochondrial ribosomal RNAs is relatively unaffected by chloramphenicol and that, despite the deficiency of small subunits, 19 S and 25 S RNA are present in normal ratios in whole mitochondria. By contrast, 19 S RNA in poky mitochondria is rapidly degraded leading to a decreased ratio of 19 S to 25 S RNA. The significance of these results with respect to the etiology of the poky mutation is discussed and a model of mitochondrial ribosome assembly that incorporates all available data is presented.     

Structural Insights into ribosome recycling factor interactions with the 70S ribosome

At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.     

Surface topography of the Bacillus stearothermophilus ribosome.

Summary The surface topography of the intact 70S ribosome and free 30S and 50S subunits from Bacillus stearothermophilus strain 2184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more ¹²⁵I than did 50S subunits derived from 70S ribosomes, whereas free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of ¹²⁵I. Iodinated 70S ribosomes and subunits retained 62–78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were S4, S7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins S2, S3, S7, S13, Sa, L5/9, L10, L11 and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including S5, S9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosome than in the free subunits. The locations of tyrosine residues in some homologus ribosomal proteins from B. stearothermophilus and E. coli are compared.     

Characterization of proteins from mitochondrial ribosomes of Locusta migratoria by three different two-dimensional gel electrophoretic methods and comparison with cytoplasmic ribosomal proteins

• 1.1. Proteins were isolated from subunits of mitochondrial and cytoplasmic ribosomes of Locusta migratoria and were analyzed by means of two-dimensional gel electrophoreses using three different electrophoresis systems.
• 2.2. Using the system of Czempiel et al. (1976) proteins from whole locust mitochondrial ribosomes (combined subunits) were separated into 72 spots; proteins from the large and small subunits resulted in 48 and 29 spots respectively.
• 3.3. The mol. wt distribution of mitochondrial ribosome proteins was estimated by using the electrophoresis system of O'Farrell (1975). These mol. wts are in the range of 11,000–56,000, the average mol. wt is about 29,500. Assuming one copy of protein per ribosome this gives a total mol. wt for the protein part of mitochondrial ribosomes of ca. 2.1 x 10⁶.
• 4.4. Parallel separation of cytoplasmic and mitochondrial ribosome proteins was achieved using the system of Geyl et al. (1981). Cytoplasmic ribosome proteins produced 65 spots and revealed a more alkaline character than mitochondrial ribosome proteins.

     

The Human Mitochondrial DNA Depletion Syndrome Gene MPV17 Encodes a Non-selective Channel That Modulates Membrane Potential

The human MPV17-related mitochondrial DNA depletion syndrome is an inherited autosomal recessive disease caused by mutations in the inner mitochondrial membrane protein MPV17. Although more than 30 MPV17 gene mutations were shown to be associated with mitochondrial DNA depletion syndrome, the function of MPV17 is still unknown. Mice deficient in Mpv17 show signs of premature aging. In the present study, we used electrophysiological measurements with recombinant MPV17 to reveal that this protein forms a non-selective channel with a pore diameter of 1.8 nm and located the channel''s selectivity filter. The channel was weakly cation-selective and showed several subconductance states. Voltage-dependent gating of the channel was regulated by redox conditions and pH and was affected also in mutants mimicking a phosphorylated state. Likewise, the mitochondrial membrane potential (Δψ_m) and the cellular production of reactive oxygen species were higher in embryonic fibroblasts from Mpv17^−/− mice. However, despite the elevated Δψ_m, the Mpv17-deficient mitochondria showed signs of accelerated fission. Together, these observations uncover the role of MPV17 as a Δψ_m-modulating channel that apparently contributes to mitochondrial homeostasis under different conditions.     

Characterization of different conformational forms of 30 S ribosomal subunits in isolated and associated states: possible correlations between structure and function.

The reaction pattern with N-[¹⁴C]ethylmaleimide served to follow conformational changes of 30 S ribosomal subunits that are induced by association with 50 S subunits and by the binding of aminoacyl-tRNA to 70 S ribosomes either enzymatically or non-enzymatically.The usefulness of the reaction with N-ethylmaleimide in discerning different conformational forms of the ribosome was previously demonstrated (Ginzburg et al., 1973) in an analysis of inactive and active 30 S subunits (as obtained at low Mg²⁺ and after heat reactivation, respectively). The reaction pattern of the 30 S moiety of 70 S ribosomes differs from the pattern of isolated active subunits (the only form capable of forming 70 S ribosomes) in both the nature of the labeled proteins and in being Mg²⁺-dependent. The reaction at 10 mm-Mg²⁺ reveals the following differences between isolated and reassociated 30 S subunits: (1) proteins S1, S18 and S21 that are not labeled in isolated active subunits, but are labeled in the inactive subunits, are highly reactive in 70 S ribosomes; (2) proteins S2, S4, S12 and S17 that uniquely react with N-ethylmaleimide in active subunits are all rendered inaccessible to modification after association; and (3) proteins S9, S13 and S19, that react in both active and inactive 30 S subunits, are labeled to a lesser extent in the 70 S ribosomes than in isolated subunits. This pattern is altered in two respects when the reaction with the maleimide is carried out at 20 mm-Mg²⁺; protein S18 is not modified while S17 becomes labeled.The differences in reaction pattern are considered as manifesting the existence of different conformational forms of the 30 S subunit in the dissociated and associated states as well as of different forms of 70 S ribosomes. The 30 S moiety of 70 S ribosomes at 10 mm-Mg²⁺ resembles the inactive subunit, while some of the features of the active subunit are preserved in the 70 S ribosome at 20 mmMg²⁺. The structural changes appear to be expressed in the functioning of the ribosome: non-enzymatic binding of aminoacyl-tRNA to active 30 S subunits is suppressed by 50 S subunits at 10 mm but not at 20 mm-Mg²⁺ (Kaufmann &; Zamir, 1972). The fact that elongation factor Tu-mediated binding is not suppressed by 50 S subunits raises the possibility that the function of the elongation factor might involve the facilitation of a conformational change of the ribosome. The analysis of different ribosomal binding complexes with N-ethylmaleimide showed that the binding of poly(U) alone results in a decrease in the labeling of S1 and S18. Binding of aminoacyl-tRNA, on the other hand, is closely correlated with the exposure of S17 for reaction with the maleimide. A model is outlined that accounts for this correlation as well as for the proposed role of elongation factor Tu.     

Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA

In trans-translation transfer messenger RNA (tmRNA) and small protein B (SmpB) rescue ribosomes stalled on truncated or in other ways problematic mRNAs. SmpB promotes the binding of tmRNA to the ribosome but there is uncertainty about the number of participating SmpB molecules as well as their ribosomal location. Here, the interaction of SmpB with ribosomal subunits and ribosomes was studied by isolation of SmpB containing complexes followed by chemical modification of ribosomal RNA with dimethyl sulfate, kethoxal and hydroxyl radicals. The results show that SmpB binds 30S and 50S subunits with 1:1 molar ratios and the 70S ribosome with 2:1 molar ratio. SmpB-footprints are similar on subunits and the ribosome. In the 30S subunit, SmpB footprints nucleotides that are in the vicinity of the P-site facing the E-site, and in the 50S subunit SmpB footprints nucleotides that are located below the L7/L12 stalk in the 3D structure of the ribosome. Based on these results, we suggest a mechanism where two molecules of SmpB interact with tmRNA and the ribosome during trans-translation. The first SmpB molecule binds near the factor-binding site on the 50S subunit helping tmRNA accommodation on the ribosome, whereas the second SmpB molecule may functionally substitute for a missing anticodon stem–loop in tmRNA during later steps of trans-translation.     

Oat seed globulin: subunit characterization and demonstration of its synthesis as a precursor

The predominant storage protein of oat (Avena sativa L.) seeds is a saline-soluble globulin with a mol wt of 320,000 which is composed of six large (M_r = 35,000 to 40,000) and six small (M_r = 20,000 to 25,000) subunits. Experiments were conducted to further describe the subunit polypeptides and to identify the initial translation products of globulin mRNAs. Approximately 20 large subunits and 10 small subunits were resolved by two-dimensional gel analysis. The large and small subunits had acidic and basic isoelectric points, respectively. Disulfide-linked complexes of one large and one small subunit were isolated by extraction in buffer lacking a reducing agent. The NH₂-terminal sequence of the small subunits was homologous to a small subunit of soybean glycinin. Immunoprecipitation of in vitro translation products of poly(A)⁺ RNA with anti-oat globulin sera yielded M_r = 60,000 to 68,000 polypeptides. In vivo labeling of spikelets with radioactive amino acids resulted in high amounts of incorporation into polypeptides with M_r = 65,000 to 68,000 which were immunoprecipitated with anti-globulin sera. These two results suggest oat globulin is synthesized as a higher mol wt precursor which is subsequently processed to yield the large and small subunit polypeptides.     

Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly

Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including MTERF4:NSUN4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.     

Functional Interaction between Ribosomal Protein L6 and RbgA during Ribosome Assembly

RbgA is an essential GTPase that participates in the assembly of the large ribosomal subunit in Bacillus subtilis and its homologs are implicated in mitochondrial and eukaryotic large subunit assembly. How RbgA functions in this process is still poorly understood. To gain insight into the function of RbgA we isolated suppressor mutations that partially restored the growth of an RbgA mutation (RbgA-F6A) that caused a severe growth defect. Analysis of these suppressors identified mutations in rplF, encoding ribosomal protein L6. The suppressor strains all accumulated a novel ribosome intermediate that migrates at 44S in sucrose gradients. All of the mutations cluster in a region of L6 that is in close contact with helix 97 of the 23S rRNA. In vitro maturation assays indicate that the L6 substitutions allow the defective RbgA-F6A protein to function more effectively in ribosome maturation. Our results suggest that RbgA functions to properly position L6 on the ribosome, prior to the incorporation of L16 and other late assembly proteins.     

C7orf30 is necessary for biogenesis of the large subunit of the mitochondrial ribosome

Defects of the translation apparatus in human mitochondria are known to cause disease, yet details of how protein synthesis is regulated in this organelle remain to be unveiled. Here, we characterize a novel human protein, C7orf30 that contributes critically to mitochondrial translation and specifically associates with the large subunit of the mitochondrial ribosome (mt-LSU). Inactivation of C7orf30 in human cells by RNA interference results in respiratory incompetence owing to reduced mitochondrial translation rates without any appreciable effects on the steady-state levels of mitochondrial mRNAs and rRNAs. Ineffective translation in C7orf30-depleted cells or cells overexpressing a dominant-negative mutant of the protein results from aberrant assembly of mt-LSU and consequently reduced formation of the monosome. These findings lead us to propose that C7orf30 is a human assembly and/or stability factor involved in the biogenesis of the large subunit of the mitochondrial ribosome.     

A novel GTPase activated by the small subunit of ribosome

The GTPase activity of Escherichia coli YjeQ, here named RsgA (ribosome small subunit-dependent GTPase A), has been shown to be significantly enhanced by ribosome or its small subunit. The enhancement of GTPase activity was inhibited by several aminoglycosides bound at the A site of the small subunit, but not by a P site-specific antibiotic. RsgA stably bound the small subunit in the presence of GDPNP, but not in the presence of GTP or GDP, to dissociate ribosome into subunits. Disruption of the gene for RsgA from the genome affected the growth of the cells, which predominantly contained the dissociated subunits having only a weak activation activity of RsgA. We also found that 17S RNA, a putative precursor of 16S rRNA, was contained in the small subunit of the ribosome from the RsgA-deletion strain. RsgA is a novel GTPase that might provide a new insight into the function of ribosome.     

Uniparental isodisomy of chromosome 2 causing MRPL44-related multisystem mitochondrial disease

Mutations in nuclear-encoded protein subunits of the mitochondrial ribosome are an increasingly recognised cause of oxidative phosphorylation system (OXPHOS) disorders. Among them, mutations in the MRPL44 gene, encoding a structural protein of the large subunit of the mitochondrial ribosome, have been identified in four patients with OXPHOS defects and early-onset hypertrophic cardiomyopathy with or without additional clinical features. A 23-year-old individual with cardiac and skeletal myopathy, neurological involvement, and combined deficiency of OXPHOS complexes in skeletal muscle was clinically and genetically investigated. Analysis of whole-exome sequencing data revealed a homozygous mutation in MRPL44 (c.467 T?>?G), which was not present in the biological father, and a region of homozygosity involving most of chromosome 2, raising the possibility of uniparental disomy. Short-tandem repeat and genome-wide SNP microarray analyses of the family trio confirmed complete maternal uniparental isodisomy of chromosome 2. Mitochondrial ribosome assembly and mitochondrial translation were assessed in patient derived-fibroblasts. These studies confirmed that c.467 T?>?G affects the stability or assembly of the large subunit of the mitochondrial ribosome, leading to impaired mitochondrial protein synthesis and decreased levels of multiple OXPHOS components. This study provides evidence of complete maternal uniparental isodisomy of chromosome 2 in a patient with MRPL44-related disease, and confirms that MRLP44 mutations cause a mitochondrial translation defect that may present as a multisystem disorder with neurological involvement.

     

Three-dimensional structure of the mammalian cytoplasmic ribosome

A three-dimensional reconstruction of the 80 S ribosome from rabbit reticulocytes has been calculated from low-dose electron micrographs of a negatively stained single-particle specimen. At 37 A resolution, the precise orientations of the 40 S and 60 S subunits within the monosome can be discerned. The translational domain centered on the upper portion of the subunit/subunit interface is quite open, allowing considerable space between the subunits for interactions with the non-ribosomal macromolecules involved in protein synthesis. Further, the cytosolic side of the monosome is strikingly more open than the membrane-attachment side, suggesting a greater ease of communication with the cytoplasm, which would facilitate the inwards and outwards diffusion of a number of ligands. Although the 60 S subunit portion of the 80 S structure shows essentially all of the major morphological features identified for the eubacterial 50 S large subunit, it appears to possess a region of additional mass that evidently accounts for the more ellipsoidal form of the eukaryotic subunit.     

Mutants of Escherichia coli lacking ribosomal protein L1

Two independently isolated mutants of Escherichia coli, RD19 and MV17-10, that appeared to lack protein L1 on their ribosomes, as determined by two-dimensional gels, were subjected to a battery of immunological tests to find if L1 was indeed lacking. The tests involved Ouchterlony double diffusion, modified immunoelectrophoresis, dimer formation on sucrose gradients, and affinity chromatography. By all these criteria, protein L1 was missing from the ribosome in these mutants. Nor was any L1 cross-reacting material detectable in the supernatant. There was, however, a specific two- to fivefold increase in concentrations of protein L11 in the supernatants of the mutants, which was evidence that protein L1 acts as a feedback inhibitor of expression of the operon coding for the genes for proteins L11 and L1.Electron micrographs of ribosomes obtained from these mutants were indistinguishable from those of wild-type strains. 50 S ribosomal subunits from mutants RD19 and MV17-10 were reconstituted with purified L1 from wild-type and investigated by immunoelectron microscopy. The three-dimensional location of ribosomal protein L1 on the surface of the large subunit was determined. L1 is located on the wider lateral protuberance of the 50 S subunit. The position of protein L1 in 50 S subunits reconstituted from mutants RD19 and MV17-10 was indistinguishable from the position in subunits from wild-type.     

Inhibition of peptide chain termination by antibodies specific for ribosomal proteins

The effects of antibodies specific for the Escherichia coli 30 S and 50 S ribosomal proteins have been determined for in vitro peptide chain termination and two partial reactions, the codon-directed binding of E. coli release factor to the ribosome and peptidyl-tRNA hydrolysis with RF². Antibodies to ribosomal proteins L7 and L12 inhibit the initial binding of RF to the ribosome, and as a result, the subsequent peptidyl-tRNA hydrolysis. The kinetics of ribosomal inactivation for in vitro termination by anti-L7/L12 indicate that Fab fragments bind to three ribosome sites, and suggest that each of three copies of L7/L12 is involved in the binding of RF to the ribosome. When 70 S ribosome substrates are pretreated with anti-L11 and anti-L16 RF-dependent peptidyl-tRNA, hydrolysis is partially inhibited but the interaction of RF with the ribosome is not affected. The inactivation of in vitro termination by a mixture of anti-L11 and anti-L16 is not co-operative. Pretreatment of the 30 S ribosomal subunit (but not 70 S ribosomal substrate) with antibodies to the 30 S proteins, S9 and S11, results in strong inhibition of codon-directed hydrolysis of peptidyl-tRNA. While these antibodies inhibit ribosome subunit association, a requirement for peptide chain termination, and thereby may inhibit the in vitro termination reactions indirectly, the codon-directed binding of RF is markedly more affected than peptidyl-tRNA hydrolysis by anti-S9 and anti-S11. Antibody to S2 and anti-S3 exhibit a similar but less marked differential effect on the partial reactions of in vitro termination under the same conditions. When dissociated ribosomes are pretreated with anti-L11, in vitro termination is completely inhibited and both codon-directed binding of RF and peptidyl-tRNA hydrolysis are affected. L11 may, therefore, be at or near the interface between the ribosome subunits and like S9 and S11 not completely accessible to antibody in 70 S ribosomes. Pretreatment of dissociated ribosomes with antibodies to a number of other ribosomal proteins (L2, L4, L6, L14, L15, L17, L18, L20, L23, L26, L27) results in partial inhibition of all termination reactions although these antibodies have no effect on termination when incubated with 70 S ribosome substrates. The antibodies probably affect in vitro termination indirectly as a result of either preventing correct ribosome subunit association, or preventing correct positioning of the fMet-tRNA at the ribosome P site.     

Intrinsic molecular properties of the protein-protein bridge facilitate ratchet-like motion of the ribosome

The ribosomal intersubunit bridges maintain the overall architecture of the ribosome and thereby play a pivotal role in the dynamics of translation. The only protein-protein bridge, b1b, is formed by the two proteins, S13 and L5 of the small and large ribosomal subunits, respectively. B1b absorbs the largest movement during ratchet-like motion, and its two proteins reorganize in different constellations during this motion of the ribosome.Our results in this study of b1b in the Escherichia coli 70S ribosome suggest that the intrinsic molecular features of the bridging proteins allow the bridge to modulate the ratchet-like motion in a controlled manner. Additionally, another large subunit protein, L31, seems to participate with S13 and L5 in the formation, dynamics, and stabilization of this bridge.