Structural dynamics of bacterial ribosomes: II. Preparation and characterization of ribosomes and subunits active in the translation of natural messenger RNA

Ribosomes from Escherichia coli were tested for activity in initiation with R17 RNA as messenger. All vacant 70 S ribosomes but not all subunits were found to be active. The ability of 30 S and 50 S subunits to form a 70 S couple at Mg²⁺ concentrations above 4 mm is a stringent test for activity.Fresh extracts, prepared at 10 mm-Mg²⁺ from cells harvested after slow cooling contain up to 80% of the ribosomes in the form of vacant 70 S couples and 20% of free subunits. The proportion of subunits increases with standing as a result of the preferential inactivation of the 50 S particles. “Native” subunits are heterogeneous and consist mostly of active 30 S and inactive 50 S particles.In contrast to 50 S subunits, 30 S subunits prepared by exposure of 70 S ribosomes to low Mg²⁺ concentrations, are largely inactive and unable to reassociate with their active 50 S counterparts. However, both initiation and association activity can be restored by heating.The results imply that the structures necessary for subunit association are most critical for the biological activity of ribosomes, presumably because they are topologically closely related to the binding sites for messenger RNA, transfer RNA, and the protein factors for initiation, translocation and termination.     

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.     

Available sulphydryl groups of mammalian ribosomes in different functional states

The numbers of sulphydryl groups on NH₄Cl-washed rat liver polyribosomes in different functional states were measured under carefully standardized conditions with ¹⁴C-labelled N-ethylmaleimide and ³⁵S-labelled 5,5-dithio-bis(2-nitrobenzoic acid). Ribosomes denatured with urea had 120 titratable sulphydryl groups, 60 on each subunit, whereas native ribosomes invariably showed fewer available sulphydryl groups. Ribosomes stripped of transfer RNA (S-type ribosomes) had 55 available sulphydryl groups. Ribosomes bearing the growing peptidyl-tRNA at the acceptor site had 41 sulphydryl groups available. If these A-type ribosomes were labelled with ¹⁴C-labelled N-ethylmaleimide and dissociated into subunits, 23 of the labelled sulphydryl groups were found on the 60 S subunit and 19 on the 40 S subunit. After translocation of the peptidyl-tRNA to the donor position on ribosomes (D ribosomes), the number of available sulphydryl groups increased to 72, of which 43 were on the 60 S subunit and 29 on the 40 S subunit. This demonstrates that both subunits participate in the change of peptidyl-tRNA from the A to D positions. When the D ribosomes were reacted with EF2 (elongation factor) and GTP, the available sulphydryl groups increased to 82; addition of EF2 alone or with GDP, GDPCP or ATP failed to cause this increase, which has accordingly been attributed to an energy-dependent conformational change in the ribosome.Ribosomes were reconstructed from subunits with poly(U) and Phe-tRNA. In the presence of poly(U) only, a ribosome with 55 available SH groups was formed, thus corresponding to the stripped ribosomes. When both poly(U) and Phe-tRNA were present, a ribosome was formed with 44 available sulphydryl groups, corresponding approximately to an A-type ribosome. Since no EF1 or GTP was used in reconstructing this ribosome, these data indicate that the conformation of A-type ribosomes is not dependent on EF1 or GTP, but is due to the presence of tRNA at the acceptor site.We therefore incline to the view that the observed changes in available SH groups reflect conformational changes, with an opening up of ribosome structure as it progresses from having the peptidyl-tRNA at the A position to the D position and then binds EF2 and GTP, followed by a restoration of the more compact from when the incoming aminoacyl-tRNA is then bound.     

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.     

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.     

Structural dynamics of bacterial ribosomes. I. Characterization of vacant couples and their relation to complexed ribosomes

Ribosomes not engaged in protein synthesis (vacant couples), in contrast to complexed ribosomes bearing nascent chains, dissociate during sedimentation in sucrose gradients at high g forces and at Mg²⁺ concentrations below 15 mm. As a result of this dissociation, a new peak between the 70 S complexed ribosomes and the free 50 S subunits is observed, the position of which shifts from about 55 S to 70 S as the Mg²⁺ concentration in the gradient is raised from 5 to 15 mm. The apparent 60 S peak consists of 50 S subunits produced during dissociation in the gradient. At low g forces, the sedimentation rate of complexed and vacant ribosomes is indistinguishable, even at 5 mm-Mg²⁺. These sedimentation properties are valid criteria to differentiate vacant and complexed ribosomes. This is shown by converting complexed ribosomes quantitatively into vacant couples by removing the nascent chains through termination release or with puromycin, or by converting vacant couples into initiation complexes with R17 RNA, fMet-tRNA and initiation factors.Ribosomes from cells harvested by slow cooling consist almost entirely of vacant couples, all of which are active in protein synthesis with natural messengers. The structural features responsible for the interaction between subunits are discussed.     

Release of 70 S ribosomes from polysomes in Escherichia coli

In order to determine whether ribosomes are released from messenger RNA as intact particles or as subunits, polysomes of Escherichia coli labeled with heavy isotopes were allowed to run off together with “light” polysomes. The normally rapid post-run-off exchange of subunits by free ribosomes was virtually eliminated by two means: the use of purified polysomes (relatively free of initiation factors), and incubation at a lower temperature (25 °C), or at a somewhat higher Mg²⁺ concentration (12 to 14 mm), than is conventional. Under these conditions ribosomes released by run-off or by puromycin accumulated without subunit exchange. Hence, even though the ribosome normally initiates via subunits, it is released from RNA by a conformational change in the intact 70 S particle, rather than by dissociation.     

Protein-protein proximity in the association of ribosomal subunits of Escherichia coli: crosslinking of 30 S protein S16 to 50 S proteins by glutaraldehyde or formaldehyde

The interaction of ribosomal subunits from Escherichia coli has been studied using crosslinking reagents. Radioactive ³⁵S-labeled 50 S subunits and non-radioactive 30 S subunits were allowed to reassociate to form 70 S ribosomes. The 70 S particles, containing radioactivity only in the 50 S protein moiety, were incubated with glutaraldehyde or formaldehyde. As a result of this treatment a substantial fraction of the 70 S particles did not dissociate at 1 mm-Mg²⁺. This fraction was isolated and the ribosomal proteins were extracted. The protein mixture was analyzed by the Ouchterlony double diffusion technique by using eighteen antisera prepared against single 30 S ribosomal proteins (all except those against S3, S15 and S17). As a result of the crosslinking procedure it was found that only anti-S16 co-precipitated ³⁵S-labeled 50 S protein. It is concluded that the 30 S protein S16 is at or near the site of interaction between subunits and can become crosslinked to one or more 50 S ribosomal proteins.     

Elongation factor Tu ternary complex binds to small ribosomal subunits in a functionally active state

A complex between elongation factor Tu (EF-Tu), GTP, phenylalanyl-tRNA (Phe-tRNA), oligo(uridylic acid) [oligo(U)], and the 30S ribosomal subunit of Escherichia coli has been formed and isolated. Binding of the EF-Tu complex appears to be at the functionally active 30S site, by all biochemical criteria that were examined. The complex can be isolated with 0.25-0.5 copy of EF-Tu bound per ribosome. The binding is dependent upon the presence of both the aminoacyl-tRNA and the cognate messenger RNA. Addition of 50S subunits to the preformed 30S-EF-Tu-GTP-Phe-tRNA-oligo(U) complex ("30S-EF-Tu complex") causes a rapid hydrolysis of GTP. This hydrolysis is coordinated with the formation of 70S ribosomes and the release of EF-Tu. Both the release of EF-Tu and the hydrolysis of GTP are stoichiometric with the amount of added 50S subunits. 70S ribosomes, in contrast to 50S subunits, neither release EF-Tu nor rapidly hydrolyze GTP when added to the 30S-EF-Tu complexes. The inability of 70S ribosomes to react with the 30S-EF-Tu complex argues that the 30S-EF-Tu complex does not dissociate prior to reaction with the 50S subunit. The requirements of the 30S reaction for Phe-tRNA and oligo(U) and the consequences of the addition of 50S subunits resemble the reaction of EF-Tu with 70S ribosomes, although EF-Tu binding to isolated 30S subunits does not occur during the elongation microcycle. This suggests that the EF-Tu ternary complex binds to isolated 30S subunits at the same 30S site that is occupied during ternary complex interaction with the 70S ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-ethyl maleimide as a probe for the study of functional sites and conformations of 30 S ribosomal subunits

Escherichia coli 30 S ribosomal subunits are inactive in a number of specific functions when Mg²⁺ concentration is reduced to 1 mM, and activity is recovered on heating under appropriate ionic conditions. When active and inactive forms were treated with N-ethyl maleimide, both forms reacted to a similar extent, but the reagent attached mostly to different proteins. Moreover, it caused irreversible inactivation only when reacting with the inactive form of the subunit. Though the activating treatment failed to restore activity to these subunits it did expose the same sulfhydryl groups as are available in the active state for reaction with the maleimide.Different ribosomal activities were eliminated at different maleimide concentrations, permitting the assignment of specific functions to sulfhydryl groups of specific ribosomal proteins. Protein S18 appears to be involved in subunit association, binding of fMet-tRNA and of aminoacyl-tRNA to the P-site. Proteins S1, S14 and S21 are all or in part involved in the binding of aminoacyl-tRNA to the A-site and in the binding of the antibiotic dihydrostreptomycin.The reaction with N-ethyl maleimide thus provides a criterion other than biological activity for characterizing different ribosomal forms and a tool for mapping the 30 S subunit for specific functional sites.     

The mechanism of action of initiaton factor 3 in protein synthesis,I. Studies on ribosomes crosslinked with dimethylsuberimidate

The mechanism of action of chain initiation factor 3 in translation was examined by using $\text{E. coli}$ 70S ribosomes which were covalently crosslinked with dimethylsuberimidate. Crosslinked ribosomes were inactive in AUG-dependent fMet-tRNA binding, and were not stimulated by IF-3 in poly(U) translation. IF-3 is known to be required for maximal rates of amino acid incorporation with synthetic polynucleotides at 18 mM Mg²⁺. A direct interaction of IF-3 with 70S ribosomes was demonstrated by crosslinking ¹⁴C-labeled IF-3 to 70S ribosomes. The labeled factor was also crosslinked to 30S and 50S ribosomal subunits. A model is presented proposing the mechanism of action of IF-3 on 70S ribosomes.     

Specific binding of ribosome recycling factor (RRF) with the Escherichia coli ribosomes by BIACORE

The direct assays on Biacore with immobilised RRF and purified L11 from E. coli in the flow trough have shown unspecific binding between the both proteins. The interaction of RRF with GTPase domain of E. coli ribosomes, a functionally active complex of L11 with 23S r RNA and L10.(L7/L12)₄ was studied by Biacore. In the experiments of binding of RRF with 30S, 50S and 70S ribosomes from E. coli were used the antibiotics thiostrepton, tetracycline and neomycin and factors, influencing the 70S dissociation Mg²⁺, NH₄Cl, EDTA. The binding is strongly dependent from the concentrations of RRF, Mg²⁺, NH₄Cl, EDTA and is inhibited by thiostrepton. The effect is most specific for 50S subunits and indicates that the GTPase centre can be considered as a possible site of interaction of RRF with the ribosome. We can consider an electrostatic character of the interactions with most probable candidate 16S and 23S r RNA at the interface of 30S and 50S ribosomal subunits.     

Role of polyamines in the binding of initiator tRNA to the 70S ribosomes of extreme thermophilic bacterium Calderobacterium hydrogenophilum

Slowly cooled cells of an extreme thermophilic eubacterium Calderobacterium hydrogenophilum possess ribosomes with weakly associated subunits. These ribosomal subunits are capable of association to 70S ribosomes either at higher Mg²⁺ concentrations (30–40 mM) or at 4–10 mM Mg²⁺ and in the presence of polyamines. The contribution of 30S and 50S subunits to the hydrodynamic stability of ribosomes was examined by forming hybrid 30S–50S couples from C. hydrogenophilum and Escherichia coli. At lower Mg²⁺ (4–10 mM) heterogeneous subunits containing 30S E. coli and 50S C. hydrogenophilum and homogeneous subunits of the thermophilic bacterium associated only in the presence of polyamines. Ribosomal subunits associated at 30 mM Mg²⁺ lose thermal stability and activity concerning poly(AUG)-dependent binding of f[³H]Met-tRNA to the P-site on 70S ribosomes or translation of poly(UG). Poly(AUG), deacylated-tRNA or initiator-tRNA have no valuable effect on association of 30S and 50S subunits. Protein synthesis initiation factor IF3 of C. hydrogenophilum prevents association of ribosomal subunits to 70S ribosomes at physiological temperature (70°C). The factor also stimulates dissociation of 70S ribosomes of E. coli at 37°C. The codon-specific binding of f[³H]Met-tRNA to homogeneous 70S ribosomes of C. hydrogenophilum at 70°C is dependent on the presence of initiation factors and concentrations of tri-pentaamines. However, excess of polyamines inhibited the reaction. Our results indicate that tri-pentaamines enhance conformational stability of 70S initiation complex at elevated temperatures.     

A fluorescent derivative of ribosomal protein S18 which permits direct observation of messenger RNA binding.

70 S Escherichia coli ribosomes were reacted with the fluorescent dye N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid for 10 min under mild conditions. The resulting ribosomes were fully active. 30 S subunits isolated from these particles were also fully active. They contain approximately 0.7 eq of fluorescent dye. Nearly all of it is attached to protein S18. Competitive reaction with N-ethylmaleimide implies that the fluorescent dye is located at cysteine 10 of the protein. The labeled 30 S particles will recombine with 50 S subunits to form stable 70 S particles. Thus the procedures we have developed allow the large scale preparation of an active fluorescent conjugate of the 70 S ribosome. The fluorescence of the 70 S particles is sensitive to the binding of mRNA, showing both quenching and a shift in emission spectra. Thus it affords a simple way to quantitate mRNA binding directly. In pilot studies without tRNA, the binding constant of the initiation triplet codon adenylyl-(3' leads to 5')-uridylyl-(3' leads to 5')-guanosine to 70 S ribosome was found to be an order of magnitude larger than that of polyuridylic acid.     

Aminoacyl-transfer RNA binding to active 30 s subunits: Effect of 50 s subunits and a new role for elongation factor T

It has been formerly shown that low concentrations of Mg²⁺, as are used to dissociate ribosomes into subunits, render the ribosomes inactive in binding of aminoacyl-transfer RNA. Activity is restored on heating the ribosomes under appropriate salt conditions. We have now studied the binding of aminoacyl-tRNA to fully activated ribosomes thus eliminating any possible activation during the binding reaction. The requirements and kinetics of the reaction as revealed are therefore those of the binding reaction and not of the activation step. The major findings were as follows.

1.

(i) Non-enzymic binding of Phe-tRNA to 30 s subunits proceeds efficiently in the cold and at Mg²⁺ concentrations as low as 5 mM.     

A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit

Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg²⁺ exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change.     

Alteration of the structure of theEscherichia coli ribosomes on treatment with Fab fragment of immunoglobulin raised against the ribosomes

Rabbits were immunised againstEscherichia coli ribosomes and the partially purified immunoglobulin G fraction had maximum ability to precipitate the ribosomes as well as the extracted ribosomal proteins. By digestion of immuno-globulin G with papain, monovalent Fab fragments were produced. The 70 S ribosome and its subunits (50 S and 30 S) were separately treated with Fab and then tested in the kinetic assay of degradation of ribosomes by ribonuclease I at various Mg²⁺ concentrations. Treated ribosomes and their subunits were degraded at faster rates than the nontreated ones; the rates in both the control and the treated cases were dependent on the concentration of Mg²⁺. These results indicate the unfolding of the structure of the ribosome on treatment with antibody fragments, which may be due to the weakening of the interaction between rRNAs and ribosomal proteins.     

Studies of Ribosomal Diffusion Coefficients Using Laser Light-Scattering Spectroscopy

Using an optical beating technique, the diffusion coefficients and relative scattered intensity of Escherichia coli 70S, 50S, and 30S ribosomes are measured as a function of temperature and Mg²⁺ concentration. For solutions at 10 mM Mg²⁺ and between 0°C and about 40°C, the values of D_20,w obtained are 1.7, 1.9, and ≈2.1 × 10^-7 cm²/s, respectively. Preparative procedures drastically affect these values and equivalent hydrodynamic ellipsoids of revolution models give large axial ratios indicating extensive hydration or a deviation from the assumed shape. Calculations also indicate that the subunits expand upon dissociation. Measurements of D_20,w vs. temperature indicate that 70S particles undergo a conformational change prior to dissociation and can be heat dissociated at 30-32°C at low concentrations. Treatment of 70S ribosomes with EDTA causes a biphasic dissociation reaction. Addition of Mg²⁺ after dissociation with EDTA shows that longer waiting times yield fewer 70S particles and that even short waiting times may yield ribosomes differing from the native conformation. Addition of p-chloromercuribenzoic acid (PCMB) is shown to dissociate 70S particles, but to a lesser extent than ethylenediaminetetraacetic acid (EDTA).     

Nondiscrimination of RNA viral message in binding to 30S ribosomes derived from T4 phage infected Escherichia coli

The effect of T4 phage on ribosomes in terms of their ability to bind RNA viral template is examined. It is found that the 30S subunits of T4 ribosomes bind MS2 RNA as efficiently as do the subunits of uninfected $\text{E. coli}$ ribosomes. On the other hand, analyses of the formation of 70S initiation complex, presumably from MS2 RNA-30S ribosome complex, using both labeled MS2 RNA and initiator tRNA, reveal that T4 ribosomes are only about half as active as $\text{E. coli}$ ribosomes. The latter phenomenon has been reported previously. These results suggest that, following T4 infection, ribosomes are modified in such a way that the attachment of fMet-tRNA_f to MS2 RNA-30S subunit complex is impaired.     

Ribosome activity and modification of 16S RNA are influenced by deletion of ribosomal protein S20

A spontaneous mutant of Escherichia coli K-12 was isolated that shows an increased misreading ability of all three nonsense codons together with an inability to grow at 42° C. It is demonstrated that the mutation is a deletion of the gene rpsT, coding for ribosomal protein S20. The loss of this protein not only influences the decoding properties of the ribosome; the modification pattern of 16S ribosomal RNA is also changed. This leads to a deficiency in the ability of the mutant to associate its 30S subunits with 50S subunits to form 70S ribosomes. It is suggested that two modified bases, m⁵C and m⁶₂A, are directly or indirectly essential for association of subunits to functional ribosomes in the rpsT mutant strain. Two other modifications were also studied; m²G which is not affected at all and m³U which is undermodified in both active and inactive subunits and, therefore, not involved in subunit association.