Letter: Conformational changes in ribosomal subunits following dissociation of the Escherichia coli 70 S ribosome

The reactivity of various Escherichia coli ribosomal proteins with N-ethylmaleimide has been used as a probe for ribosomal topography changes during the subunit-70 S transition. With the 70 S ribosome there are several proteins from both subunits which do not react with N-ethylmaleimide, but which do so after dissociation of the 70 S particle to free 30 S and 50 S subunits. The kinetics of their exposure is slow relative to that of the 70 S dissociation reaction suggesting conformational changes in both subunits subsequent to 70 S particle dissociation.     

POPS: A fast algorithm for solvent accessible surface areas at atomic and residue level

POPS (Parameter OPtimsed Surfaces) is a new method to calculate solvent accessible surface areas, which is based on an empirically parameterisable analytical formula and fast to compute. Atomic and residue areas (the latter represented by a single sphere centered on the C(alpha) atom of amino acids and at the P atom of nucleotides) have been optimised versus accurate all-atom methods. The parameterisation has been derived from a selected dataset of proteins and nucleic acids of different sizes and topologies. The residue based approach POPS-R, has been devised as a useful tool for the analysis of large macromolecular assemblies like the ribosome and it is specially suited for the refinement of low resolution structures. POPS-R also allows for estimates of the loss of free energy of solvation upon complex formation, which should be particularly useful for the design of new protein-protein and protein-nucleic acid complexes. The program POPS is available at http://mathbio.nimr.mrc.ac.uk/~ffranca/POPS and at the mirror site http://www.cs.vu.nl/~ibivu/programs/popswww.     

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.     

Ribosomal Intersubunit Bridge B2a Is Involved in Factor-Dependent Translation Initiation and Translational Processivity

Intersubunit bridges are important for holding together subunits in the 70S ribosome. Moreover, a number of intersubunit bridges have a role in modulating the activity of the ribosome during translation. Ribosomal intersubunit bridge B2a is formed by the interaction between the conserved 23S rRNA helix-loop 69 (H69) and the top of the 16S rRNA helix 44. Within the 70S ribosome, bridge B2a contacts translation factors and the A-site tRNA. In addition to bridging the subunits, bridge B2a has been invoked in a number of other ribosomal functions from initiation to termination. In the present work, single-nucleotide substitutions were inserted at positions 1912 and 1919 of Escherichia coli 23S rRNA (helix 69), which are involved in important intrahelical and intersubunit tertiary interactions in bridge B2a. The resulting ribosomes had a severely reduced activity in a cell-free translation elongation assay, but displayed a nearly wild-type-level peptidyl transferase activity. In vitro reassociation efficiency decreased with all of the H69 variant 50S subunits, but was severest with the A1919C and ΔH69 variants. The mutations strongly affected initiation-factor-dependent 70S initiation complex formation, but exhibited a minor effect on the nonenzymatic initiation process. The mutations decreased ribosomal processivity in vitro and caused a progressive depletion of 50S subunits in polysomal fractions in vivo. Mutations at position 1919 decreased the stability of a dipeptidyl-tRNA in the A-site, whereas the binding of the dipeptidyl-tRNA was rendered more stable with 1912 and ΔH69 mutations. Our results suggest that the H69 of 23S rRNA functions as a control element during enzymatic steps of translation.     

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.     

In vitro protein folding by E. coli ribosome: unfolded protein splitting 70S to interact with 50S subunit

Folding of unfolded protein on Escherichia coli 70S ribosome is accompanied by rapid dissociation of the ribosome into 50S and 30S subunits. The dissociation rate of 70S ribosome with unfolded protein is much faster than that caused by combined effect of translation and polypeptide release factors known to be involved in the dissociation of ribosome into subunits. The protein then reaches a “folding competent” state on 50S and is released to take up native conformation by itself. Release before attaining the folding competent state or prevention of release by cross-linking it with ribosome, would not allow the protein to get back to its native conformation.     

Streptomycin-induced formation of 70-S monosomes and oligomers in Chlamydomonas reinhardi

Under ionic conditions, where the 70 S ribosomes but not the 80 S ribosomes partly dissociate into the subunits, in three mutants of Chlamydomonas reinhardi streptomycin causes in vivo at first an increase, later a decrease of the 70 S ribosome fraction. This behaviour can be explained, if streptomycin acts on the ribosome cycle of the organelle ribosomes of eukaryotes in the same way as on the ribosome cycle of E. coli.Streptomycin also induces the formation of dimers and oligomers from 80 S cytoplasmic ribosomes. The kinetics of this formation is similar to that of the 70 S ribosomes. However, this effect of streptomycin does not seem to influence the functional capacity of the 80 S ribosomes.     

Topography of 16 S RNA in 30 S subunits and 70 S ribosomes accessibility to cobra venom ribonuclease

A ribonuclease extracted from the venom of the cobra Naja oxiana, which shows an unusual specificity for double-stranded RNA regions, was used to obtain new insight on the topography of Escherichia coli ribosomal 16 S RNA in the 30 S subunit and in the 70 S couple. ³²P-labeled 30 S subunits or reconstituted 70 S tight couples containing ³²P-labeled 16 S RNA have been digested under progressively stronger conditions. The cleavage sites have been precisely localized and the chronology of the hydrolysis process studied.The enzyme cleaves the 16 S RNA within 30 S subunits at 21 different sites, which are not uniformly distributed along the molecule. These results provide valuable information on the 16 S RNA topography and evidence for secondary structure features.The binding of the 50 S subunit markedly reduces the rate of the 16 S RNA hydrolysis and provides protection for several cleavage sites. Four of them are clustered in the 3′-terminal 200 nucleotides of the molecule, one in the middle (at position 772) and one in the 5′ domain (at position 336). Our results provide further evidence that the 3′-terminal and central regions of the RNA chain are close to each other in the ribosome structure and lie at the interface of the two subunits. They also suggest that the 5′ domain is probably not involved exclusively in structure and assembly.     

Structural dynamics of bacterial ribosomes: IV. Classification of ribosomes by subunit interaction

Previous studies in this series (M. Noll et al., 1973a,b; Noll & Noll, 1974) have established that in Escherichia coli the ability of subunits to form vacant 70 S ribosome couples at 10 mm-Mg²⁺ is a stringent condition for activity in the translation of natural messenger (R17 RNA). The present study examines the structural basis of subunit interaction. It is found that vacant ribosome couples prepared by various methods fall into two classes, “tight” couples and “loose” couples, that differ in the affinity of their subunits for each other. Detection and separation of the two particle species is possible by ultracentrifugation. When analyzed on sucrose gradients at 6 mm-Mg²⁺ and moderate speed (30,000 revs/min), tight couples sediment as undissociated 70 S ribosomes, whereas loose couples are completely dissociated and sediment as 30 S and 50 S subunits. At 15 mm-Mg²⁺ in the gradient, both species sediment as a 70S peak. At 10 mm-Mg²⁺ and 60,000 revs/min, two peaks (63 S and 55 S) are seen because the high hydrostatic pressure causes more pronounced dissociation of the loose than of the tight couples.Association is dependent on the state of each subunit. Removal of Mg²⁺ produces 30 S b-particles that are unable to associate with 50 S subunits unless reconverted to the 30 S a-form by thermal activation according to Zamir et al. (1971). In the dissociated state, 50 S subunits tend to change irreversibly to a 50 S b-modification that produces loose couples upon association with 30 S a-subunits. The 50 S a → 50 S b transition could not be related to breaks in 23 S RNA detectable by sedimentation analysis. However, mild treatment of 50 S a-subunits with RNase produces particles that associate with 30 S a-subunits to couples that are less stable than the loose couples resulting from a dissociation/association step.Fresh S-30 extracts contain only tight couples (approx. 80%) and subunits (approx. 20%). Our results suggest that loose couples are artefacts derived from tight couples by a structural or conformational modification.Interaction-free subunits that previously were found to form a primitive initiation complex with poly(U) and tRNA_Phe (Schreier & Noll, 1970,1971), and to be active in phenylalanine polymerization, are shown to consist of the b-form of each subunit.It is likely that conflicting results obtained in the study of the mechanism of initiation and other aspects of ribosome function are due to the lack of structural criteria required for standardizing the ribosome preparation used by different investigators. This study provides simple methods and criteria to classify and separate physically all ribosome and ribosome subunits that have been observed into well-defined classes of predictable activity.     

Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome

During protein synthesis, ribosomes become stalled on polyproline-containing sequences, unless they are rescued in archaea and eukaryotes by the initiation factor 5A (a/eIF-5A) and in bacteria by the homologous protein EF-P. While a structure of EF-P bound to the 70S ribosome exists, structural insight into eIF-5A on the 80S ribosome has been lacking. Here we present a cryo-electron microscopy reconstruction of eIF-5A bound to the yeast 80S ribosome at 3.9 Å resolution. The structure reveals that the unique and functionally essential post-translational hypusine modification reaches toward the peptidyltransferase center of the ribosome, where the hypusine moiety contacts A76 of the CCA-end of the P-site tRNA. These findings would support a model whereby eIF-5A stimulates peptide bond formation on polyproline-stalled ribosomes by stabilizing and orienting the CCA-end of the P-tRNA, rather than by directly contributing to the catalysis.     

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.     

Contribution of ribosomal residues to P-site tRNA binding

Structural studies have revealed multiple contacts between the ribosomal P site and tRNA, but how these contacts contribute to P-tRNA binding remains unclear. In this study, the effects of ribosomal mutations on the dissociation rate (k_off) of various tRNAs from the P site were measured. Mutation of the 30S P site destabilized tRNAs to various degrees, depending on the mutation and the species of tRNA. These data support the idea that ribosome-tRNA interactions are idiosyncratically tuned to ensure stable binding of all tRNA species. Unlike deacylated elongator tRNAs, N-acetyl-aminoacyl-tRNAs and tRNA^fMet dissociated from the P site at a similar low rate, even in the presence of various P-site mutations. These data provide evidence for a stability threshold for P-tRNA binding and suggest that ribosome-tRNA^fMet interactions are uniquely tuned for tight binding. The effects of 16S rRNA mutation G1338U were suppressed by 50S E-site mutation C2394A, suggesting that G1338 is particularly important for stabilizing tRNA in the P/E site. Finally, mutation C2394A or the presence of an N-acetyl-aminoacyl group slowed the association rate (k_on) of tRNA dramatically, suggesting that deacylated tRNA binds the P site of the ribosome via the E site.     

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.     

RimM and RbfA Are Essential for Efficient Processing of 16S rRNA in Escherichia coli

The trmD operon is located at 56.7 min on the genetic map of the Escherichia coli chromosome and contains the genes for ribosomal protein (r-protein) S16, a 21-kDa protein (RimM, formerly called 21K), the tRNA (m¹G37)methyltransferase (TrmD), and r-protein L19, in that order. Previously, we have shown that strains from which the rimM gene has been deleted have a sevenfold-reduced growth rate and a reduced translational efficiency. The slow growth and translational deficiency were found to be partly suppressed by mutations in rpsM, which encodes r-protein S13. Further, the RimM protein was shown to have affinity for free ribosomal 30S subunits but not for 30S subunits in the 70S ribosomes. Here we have isolated several new suppressor mutations, most of which seem to be located close to or within the nusA operon at 68.9 min on the chromosome. For at least one of these mutations, increased expression of the ribosome binding factor RbfA is responsible for the suppression of the slow growth and translational deficiency of a ΔrimM mutant. Further, the RimM and RbfA proteins were found to be essential for efficient processing of 16S rRNA.     

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.     

Reaction of ribosomal sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid)

The number of sulfhydryl groups in the Escherichia coli ribosome has been measured by titration with 5,5′-dithiobis(2-nitrobenzoic acid). Under denaturing conditions, there are 38.8 ± 1.0 titratable thiols per 70 S ribosome and 22.8 ± 0.3 and 12.9 ± 0.3 titratable thiols per 50 S and 30 S subunits, respectively. Three categories of thiol groups can be distinguished in the native 70 S ribosome, a “fast reacting” class of about 3 residues, a “slow reacting” class of about 10 residues and a “buried” class including about 26 residues. The addition of polyuridylic acid to reaction mixtures protects a fast-reacting thiol in the 30 S subunit belonging to protein S1.The addition of urea to ribosome solutions makes the buried residues titratable. Denaturation occurs as a sharp transition at a urea concentration between 4 and 4.5 m. Urea does not fully dissociate the ribosome into RNA and protein. Instead, in the case of the 30 S subunit, a slowly sedimenting particle forms in the presence of urea, containing roughly 65% of the normal amount of protein.     

Synthetic Lethality in the Tobacco Plastid Ribosome and Its Rescue at Elevated Growth Temperatures

Consistent with their origin from cyanobacteria, plastids (chloroplasts) perform protein biosynthesis on bacterial-type 70S ribosomes. The plastid genomes of seed plants contain a conserved set of ribosomal protein genes. Three of these have proven to be nonessential for translation and, thus, for cellular viability: rps15, rpl33, and rpl36. To help define the minimum ribosome, here, we examined whether more than one of these nonessential plastid ribosomal proteins can be removed from the 70S ribosome. To that end, we constructed all possible double knockouts for the S15, L33, and L36 ribosomal proteins by stable transformation of the tobacco (Nicotiana tabacum) plastid genome. We find that, although S15 and L33 function in different ribosomal particles (30S and 50S, respectively), their combined deletion from the plastid genome results in synthetic lethality under autotrophic conditions. Interestingly, the lethality can be overcome by growth under elevated temperatures due to an improved efficiency of plastid ribosome biogenesis. Our results reveal functional interactions between protein and RNA components of the 70S ribosome and uncover the interdependence of the biogenesis of the two ribosomal subunits. In addition, our findings suggest that defining a minimal set of plastid genes may prove more complex than generally believed.     

The use of cross-linking platinum reagents in the study of tRNA and mRNA interaction with ribosomes

The use of some bifunctional Pt(II)-containing cross-linking reagents for investigation of structural organization of ribosomal tRNA- and mRNA-binding centres is demonstrated for various types of [70S ribosome.mRNA-tRNA] complexes. It is shown that treatment of the complexes [70S ribosome.Ac[14C]Phe-tRNA(Phe).poly(U)], [70S ribosome.3'-32pCp-tRNA(Phe).poly(U)] and [70S ribosome.f[35S]Met-tRNA(fMet).AUGU6] with Pt(II)-derivatives results in covalent attachment of tRNA to ribosome. AcPhe-tRNA(Phe) and 3'-pCp-tRNA(Phe) bound at the P site were found to be cross-linked preferentially to 30S subunit. fMet-tRNA(fMet) within the 70S initiation complex is cross-linked to both ribosome subunits approximately in the same extent, which exceeds two-fold the level of the tRNA(Phe) cross-linking. All used tRNA species were cross-linked in the comparable degree both to rRNA and proteins of both subunits in all types of the complexes studied. 32pAUGU6 cross-links exclusively to 30S subunit (to 16S RNA only) within [70S ribosome.32pAUGU6.fMet-tRNA(fMet)] complex. In the absence of fMet-tRNAfMet the level of the cross-linking is 4-fold lower.     

Sucrose density gradient sedimentation of E. coli ribosomes.

The behavior of E. coli ribosomes during sedimentation on sucrose gradients is predicted under a variety of conditions by computer simulations. Since numerous recent kinetic studies indicate equilibration in times short compared to the time of sedimentation, these simulations assume that the system attains local reaction equilibrium at every point in the gradient at all times. For any type of homogeneous equilibrating ribosome population, governed by a single formation constant at one atmosphere pressure for 70S couples, no more than two clearly defined zones will be resolved, although the presence of large dissociating effects due to pressure gradients in high speed experiments will spread the subunit zone. Normally the pattern will consist of a 30S zone and a so-called “70S” zone, which is in reality a mixture of 70S couples and 30S and 50S subunits in local equilibrium. The greater the dissociation into subunits, the more the “70S” zone will be slowed below the nominal rate of 70 Svedberg units. If ribosomes have been collected from the “70S” zone in several successive cycles of purification, the repeated deletion of resolved 30S subunits can result in a preparation with so large a molar excess of 50S subunits that the ensuing sucrose density gradient sedimentation pattern may exhibit a “70S” zone followed by zone of 50S subunits, insteadof a zone of 30S subunits. Our most important conclusion is that whenever a well-resolved 50S zone is present in a sucrose density gradient sedimentation experiment on E. coli ribosomes, in addition to a 30S and a “70S” zone, under conditions where ribosomes and subunits should be in reversible equilibrium, the preparation must be microheterogeneous, containing a mixture of “tight” and “loose” couples. Moreover in such cases the content of large subunits in the 50S zone must be derived entirely from “loose” couples whereas the 30S zone must contain small subunits derived from both “tight” and “loose” couples. Sedimentation patterns predicted for various mixtures of “tight” and “loose” couples display all the major characteristics of published experimental patterns for E. coli ribosomes, including the partial or complete resolution into three zones, depending on rotor velocity and level of Mg²⁺.