Mechanism of the ribosome-dependent uncoupled GTPase reaction catalyzed by polypeptide chain elongation factor G.

At low NH4-+ concentrations, 50S ribosomal subunits from E. coli were fully active in the absence of 30S ribosomal subunits, in forming a complex with the polypeptide chain elongation factor G (EF-G) and guanine nucleotide (ternary complex formation), and also in supporting EF-G dependent hydrolysis of GTP (uncoupled GTPase reaction). However, both activities were markedly inhibited on increasing the concentration of the monovalent cation, and at 160 mM NH4-+, the optimal concentration for polypeptide synthesis in a cell-free system, almost no activity was observed with 50S ribosomes alone. It was found that the inhibitory effect of NH4-+ was reversed by addition of 30S subunits. Thus, at 160 mM NH4-+, only 70S ribosomes were active in supporting the above two EF-G dependent reactions, whereas at 20 mM NH4-+, 50S ribosomes were almost as active as 70S ribosomes. Kinetic studies on inhibition by NH4-+ of the formation of 50S ribosome-EF-G-guanine nucleotide complex, indicated that the inhibition was due to reduction in the number of active 50S ribosomes which were capable of interacting with EF-G and GTP at higher concentrations of NH4-+. The inhibitory effects of NH4-+ on ternary complex formation and the uncoupled GTPase reaction were markedly influenced by temperature, and were much greater at 0 degrees than at 30 degrees. A conformational change of 50S subunits through association with 30S subunits is suggested.     

Rapid peptide bond formation on isolated 50S ribosomal subunits

The catalytic site of the ribosome, the peptidyl transferase centre, is located on the large (50S in bacteria) ribosomal subunit. On the basis of results obtained with small substrate analogues, isolated 50S subunits seem to be less active in peptide bond formation than 70S ribosomes by several orders of magnitude, suggesting that the reaction mechanisms on 50S subunits and 70S ribosomes may be different. Here we show that with full-size fMet-tRNA(fMet) and puromycin or C-puromycin as peptide donor and acceptor substrates, respectively, the reaction proceeds as rapidly on 50S subunits as on 70S ribosomes, indicating that the intrinsic activity of 50S subunits is not different from that of 70S ribosomes. The faster reaction on 50S subunits with fMet-tRNA(fMet), compared with oligonucleotide substrate analogues, suggests that full-size transfer RNA in the P site is important for maintaining the active conformation of the peptidyl transferase centre.     

Separation of ribosomal subunits of Escherichia coli by Sepharose chromatography using reverse salt gradient.

A mixture of 30 S and 50 S subunits quantitatively absorbs on a column of Sepharose--4B from the buffer: 0.02 M Tris--HCl, pH 7.5, containing 1.5 M (NH4)2SO4. During elution by reverse gradient of ammonium sulphate (1.5--0.05 M) the subunits are eluted at different salt concentrations. Complete separation of subunits is attained in the absence of Mg2+ ions. The 30 S subunits prepared from 70 S ribosomes according to this procedure are fully active in the codon--dependent binding of a specific aminoacyl--tRNA. After their reassociation with 50 S subunits isolated by zonal centrifugation, the resulting 70 S ribosomes are active in polypeptide synthesis at the same degree as control 70 S ribosomes in which both types of subunits were prepared by zonal centrifugation. The initial 70 S ribosomes for the chromatographic separation into subunits can be obtained by their pelleting from a crude extract with subsequent washing with concentrated solutions of NH4Cl in the ultracentrifuge, or by salt fractionation of the crude extract according to a slightly modified procedure of Kurland.     

Stabilization by the 30S ribosomal subunit of the interaction of 50S subunits with elongation factor G and guanine nucleotide.

The role of the 30S ribosomal subunit in the formation of the complex ribosome-guanine nucleotide-elongation factor G (EF-G) has been examined in a great variety of experimental conditions. Our results show that at a large molar excess of EF-G or high concentrations of GTP or GDP, 50S ribosomal subunits are as active alone as with 30S subunits in the formation of the complex, while at lower concentrations of nucleotide or lower amounts of EF-G, addition of the 30S subunit stimulates greatly the reaction. The presence of the 30S ribosomal subunit can also moderate the inhibition of the 50S subunit activity that occurs by increasing moderately the concentrations of K+ and NH4+, and extends upward the concentration range of these monovalent cations in which complex formation is at maximum. The Mg2+ requirement for complex formation with the 50S subunit appears to be slightly less than that needed for association of the 30S and 50S ribosomal subunits. Measurement of the reaction rate constants of the complex formation shows that the 30S ribosomal subunit has only little effect on the initial association of EF-G and guanine nucleotide with the 50S subunit; but once this complex is formed, the 30S subunit increases its stability from 10- to 18-fold. It is concluded that stabilization of the interaction between EF-G and ribosome is a major function of the 30S subunit in the ribosome-EF-G GTPase reaction.     

Puromycin binding to the small subunit of Escherichia coli ribosomes. Localization of the antibiotic in subunits reconstituted with puromycin-modified components

Small (30 S) ribosomal subunits from Escherichia coli strain TPR 201 were photoaffinity-labeled with [3H]puromycin in the presence of chloramphenicol under conditions in which more than 1 mol of antibiotic was incorporated per mol of ribosomes. The subunits were than washed with 3 M NH4Cl to yield core particles and a split protein fraction; the split proteins were further fractionated with ammonium sulfate. Subunits were then reconstituted using one fraction (core, split proteins, or ammonium sulfate supernatant) from photoaffinity-modified subunits and other components from unmodified (control) subunits. The distribution of [3H]puromycin in ribosomal proteins was monitored by one-dimensional polyacrylamide gel electrophoresis, and the sites of puromycin binding were visualized by immunoelectron microscopy. Two areas of puromycin binding were identified. A high affinity puromycin site, found on the upper third of the subunit and distant from the platform, is identical to the primary site previously identified (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894). Binding at this site is maximal in subunits reconstituted with high levels of puromycin-modified protein S14, and is decreased when unmodified S14 is incorporated. Because the percentage of antibody binding at the primary site always exceeds the percentage of puromycin label in protein S14, the primary site must include components other than S14. A secondary puromycin site of lower affinity is found on the subunit platform. This site is enriched in subunits reconstituted from puromycin-modified core particles and may include protein S7. Our results demonstrate the feasibility of localizing specifically modified components in reconstituted ribosomal subunits.     

Subunit interaction during catalysis: ammonium ion modulation of catalytic steps in the Escherichia coli glutamine synthetase reaction

A new approach for assessing if catalytic cooperativity may occur between subunits has been applied to Escherichia coli glutamine synthetase. The extent of oxygen exchange between bound [18O]glutamate and phosphate per molecule of glutamine formed was evaluated at various NH4+ concentrations. This allows calculation of the minimum number of reaction reversals in which bound glutamine is converted to bound glutamate prior to release of glutamine. At 1000 microM NH4+ no detectable reversals occurred, and only one glutamate oxygen appeared in product phosphate as required by the reaction mechanism. However, at 10 microM NH4+ over 15 reversals of bound glutamine formation occurred. Controls showed that under the experimental conditions free glutamine does not become significantly involved in exchange and, therefore, the reversal of the oxygen exchange steps appears to be limited to bound glutamine. In contrast to the effect seen with NH4+, adenosine 5'-triphosphate concentration appears to modulate the exchange of oxygen between glutamate and phosphate only slightly. These findings are interpreted as showing that NH4+ either promotes the dissociation of one of the reaction products or decreases the participation of bound products in the exchange. The NH4+ modulation of the oxygen exchange is consistent with binding of NH4+ at one catalytic site promoting catalytic events at an alternate catalytic site but does not eliminate all other explanations.     

Partial release of AcPhe-Phe-tRNA from ribosomes during poly(U)-dependent poly(Phe) synthesis and the effects of chloramphenicol

Poly(U)-programmed 70S ribosomes can be shown to be 80% to 100% active in binding the peptidyl-tRNA analogue AcPhe-tRNA to their A or P sites, respectively. Despite this fact, only a fraction of such ribosomes primed with AcPhe-tRNA participate in poly(U)-directed poly(Phe) synthesis (up to 65%) at 14 mM Mg2+ and 160 mM NH4+. Here it is demonstrated that the apparently 'inactive' ribosomes (greater than or equal to 35%) are able to participate in peptide-bond formation, but lose their nascent peptidyl-tRNA at the stage of Ac(Phe)n-tRNA, with n greater than or equal to 2. The relative loss of early peptidyl-tRNAs is largely independent of the degree of initial saturation with AcPhe-tRNA and is observed in a poly(A) system as well. This observation resolves a current controversy concerning the active fraction of ribosomes. The loss of Ac(Phe)n-tRNA is reduced but still significant if more physiological conditions for Ac(Phe)n synthesis are applied (3 mM Mg2+, 150 mM NH4+, 2 mM spermidine, 0.05 mM spermine). Chloramphenicol (0.1 mM) blocks the puromycin reaction with AcPhe-tRNA as expected but, surprisingly, does not affect the puromycin reaction with Ac(Phe)2-tRNA nor peptide bond formation between AcPhe-tRNA and Phe-tRNA. The drug facilitates the release of Ac(Phe)2-4-tRNA from ribosomes at 14 mM Mg2+ while it hardly affects the overall synthesis of poly(Phe) or poly(Lys).     

Effect of the concentration ratio of bi- and monovalent ions on the functional activity of 30S ribosome subunits of Escherichia coli

Experiments on poly(U)-dependent binding of Phe-tRNAPhe to 30S subunits revealed the existence of a critical [Mg2+]/[NH4+] ratio in a medium (approximately 0.05-0.1) with respect to the binding capacity of subunits. If the ratio is greater than the critical one, 30S subunits undergo reversible inactivation even at the highest Mg2+ concentrations (up to 20 mM). The stronger is the deviation from the [Mg2+]/[NH4+] value = 0.05-0.1, the greater are both the rate and extent of such an inactivation. Two sites for tRNA in initially active 30S subunits have been shown to be inactivated in an interdependent way. On the other hand, a progressive decrease of [Mg2+]/[NH4+] ratio in a medium (from the value of 0.05 and lower) does not produce inactivation, but rather results in reduced affinity constants of Phe-tRNAPhe for active sites of 30S subunits.     

Interaction of rat liver ribosomal subunits with homologous Met-tRNAs

Rat liver 40 S ribosomal subunits, in the presence of magnesium ions, bind homologous, resolved Met-tRNAs in the absence of added exogenous proteins. The interaction of the aminoacyl-tRNAs with the particle is dependent on the concentration of magnesium ions in the incubation. At various Mg²⁺ concentrations examined, binding of the putative initiator Met-tRNA_i to 40 S subunits is greater than that observed with Met-tRNA_m. Also, binding of Met-tRNA_i to 40 S subunits is greater than that obtained with 40 S plus 60 S particles. The initial rate of formation of the 40 S·Met-tRNA_i complex is greater at 25 °C than at 37 or 4 °C; decay of the complex, which is observed after 15 min of incubation, is greater at 37 °C but it is slower if 60 S subunits are added after the complex has been formed. If 60 S subunits are added to the incubation with 40 S subunits at the start of the reaction, binding of Met-tRNA_i is inhibited; inhibition is also obtained if elongation (binding) factor EF-1 or stripped tRNAs (particularly tRNA^Met) are present in the incubation mixture containing 40 S subunits. Acetyl-Met-tRNA_i binds to 40 S·ApUpG complex to the same extent as unacetylated Met-tRNA_i and, after addition of 60 S subunits, reacts extensively with puromycin; the addition of elongation (translocation) factor EF-2 and GTP do not affect the extent of the puromycin reaction, suggesting that the acMet-tRNA_i is bound to a site on the 40 S subunits which becomes the P site on 80 S ribosomes.     

Stimulation, by two Escherichia coli supernatant proteins, of the initiation of polypeptide synthesis.

Two protein factors (A and B) have been partially purified from Escherichia coli supernatant which, in combination, are more effective than 0.5 M NH4Cl in stimulating ribosomes for AcPhe-tRNA and fMet-tRNA binding, for the puromycin reaction, and for incorporating acetylphenylalanine from AcPhe-tRNA into polypeptide. The factors appear to differ from the initiation factors, the elongation factor EF-T, and ribosomal proteins. Some uncertainty exists as to whether factor B is different from EF-G. To maximize the effect of the factors in initiator tRNA binding, we preincubated the ribosomes with the factors and carried out the binding assay for a short period at 15 degrees C. Maximal stimulation of binding occurred after about a 2-min preincubation at 37 degrees C. Longer preincubation times were required at 15 degrees C, and only slight stimulation was observed after preincubation at 0 degrees C. The extent of stimulation by the factors was not affected when the NH4Cl concentration was increased from 40 to 500 mM in the preincubation. The presence of both the 30S and 50S ribosomal subunits is required for the enhancement of AcPhe-tRNA binding. Polyphenylalanine synthesis carried out without AcPhe-tRNA is inhibited by the factors. It is suggested that the factors may act by inducing a structural rearrangement of the ribosomes.     

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.     

Studies on the catalytic rate constant of ribosomal peptidyltransferase

A detailed kinetic analysis of a model reaction for the ribosomal peptidyltransferase is described, using fMet-tRNA or Ac-Phe-tRNA as the peptidyl donor and puromycin as the acceptor. The initiation complex (fMet-tRNA X AUG X 70 S ribosome) or (Ac-Phe-tRNA X poly(U) X 70 S ribosome) (complex C) is isolated and then reacted with excess puromycin (S) to give fMet-puromycin or Ac-Phe-puromycin. This reaction (puromycin reaction) is first order at all concentrations of S tested. An important asset of this kinetic analysis is the fact that the relationship between the first order rate constant kobs and [S] shows hyperbolic saturation and that the value of kobs at saturating [S] is a measure of the catalytic rate constant (k cat) of peptidyltransferase in the puromycin reaction. With fMet-tRNA as the donor, this kcat of peptidyltransferase is 8.3 min-1 when the 0.5 M NH4Cl ribosomal wash is present, compared to 3.8 min-1 in its absence. The kcat of peptidyltransferase is 2.0 min-1 when Ac-Phe-tRNA replaces fMet-tRNA in the presence of the ribosomal wash and decreases to 0.8 min-1 in its absence. This kinetic procedure is the best method available for evaluating changes in the activity of peptidyltransferase in vitro. The results suggest that peptidyltransferase is subjected to activation by the binding of fMet-tRNA to the 70 S initiation complex.     

Re-activation of the peptidyltransferase centre of rabbit reticulocyte ribosomes after inactivation by exposure to low concentrations of magnesium ion.

1. The larger subrivosomal particles of rabbit reticulocytes retained full activity in the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis after 4h at 0 degrees C when buffered 0.5M-NH4Cl/10-30mM-MgCl2 was the solvent. 2. Activity in the puromycin reaction was diminished to approx 10% after 15-30 min at 0 degrees C when the concentration of MgCl2 was lowered to 2mM. 3. Activity was not restored when the concentration of MgCl2 was raised from 2mM to 10-30 mM at 0 degrees C. However, activity was recovered as measured by both assay systems when the ribosome fraction was heated to 37 degrees C at the higher concentrations of MgCl2. 4. Recovery of activity was noted during the course of the polyphenylalanine synthesis in 50 mM-KCl/5mM-MgCl2/25mM-Tris/HCl, pH 7.6, at 37 degrees C. Re-activation was slow at 20 degrees C and below. 5. No more than about 5% of the protein moiety of the subparticle was lost in 0.5M-NH4Cl on decreasing MgCl2 concentration from 10mM to 2mM. No proteins were detected in the supernatant fractions by gel electrophoresis after ribosomes were separated by differential centrifugation. The supernatant fraction was not essential for the recovery of activity. However, at higher (e.g. 1M) concentrations of NH4Cl, proteins were split from the subparticle. 6. The loss and regain of activity found on lowering and restoring the concentration of MgCl2 at 0.5M-NH4Cl appears to arise from a conformational change that does not seem to be associated with a loss and regain of particular proteins. 7. A 2% decrease in E260 was noticed when the concentration of Mg2+ was restored, and the change in the spectrum indicated a net increase of approx. 100A-U base-pairs per subribosomal particle. 8. When the concentration of Mg2+ was restored, S20,W of the subparticle remained at 52+/- 1S until the sample was incubated at 37 degrees C when S20,W increased to 56 +/- 1S compared with the value of 58 +/- 1S for the subparticle as originally isolated.     

Reassembly of the peptidyltransferase centre of larger subparticles of rabbit reticulocyte ribosomes from a core-particle and split-protein fraction.

We report the reconstruction, from a core-particle and split-protein fraction, of the larger subribosomal particle of rabbit reticulocytes. The reassembled particle was active in polyphenylalanine synthesis and in the puromycin reaction. The core-particles and split-protein fractions were obtained by treatment of the larger subparticle with salt solutions containing NH4+ and Mg2+ in the molar ratio 40:1 over the range 2.25-2.75 M-NH4Cl/56-69mM-MgCl2 at 0 degrees C. This treatment led to the loss of about eight proteins (approx. 17% of the protein moiety), which were found wholly or largely in the split-protein fraction as shown by two-dimensional gel electrophoresis. The core particle retained 5S rRNA and had much decreased (no more than 10% of control) ability to function in the puromycin reaction or in poly (U)-directed polyphenylalanine synthesis. Activity was recovered when the recombined core-particle and split-protein fractions were dialysed overnight at 4 degrees C against 0.3M-NH4Cl/15mM-MgCl2/1mM-dithiothreitol/15% (v/v) glycerol/20mM-Tris/HCl, pH 7.6, and then heated for 1 h at 37 degreesEES C. The recovery was 40-80% of the original activity. Raising the concentration of MgCL2 to 300 mM in 2.5 M-NH4CL led to the removal of seven rather than eight proteins, and the core particle remained active in the puromycin reaction. We infer that the protein retained by raising the concentration of Mg2+ is an essential component of the peptidyltransferase centre of the ribosome.     

The binding of ribosomal subunits to endoplasmic reticulum membranes

The binding of ribosomes and ribosomal subunits to endoplasmic reticulum preparations of mouse liver was studied. (1) Membranes prepared from rough endoplasmic reticulum by preincubation with 0.5m-KCl and puromycin bound 60-80% of added 60S subunits and 10-15% of added 40S subunits. Membranes prepared with pyrophosphate and citrate showed less clear specificity for 60S subunits particularly when assayed at low ionic strengths. (2) Ribosomal 40S subunits bound efficiently to membranes only in the presence of 60S subunits. The reconstituted membrane-60S subunit-40S subunit complex was active in synthesis of peptide bonds. (3) No differences in binding to membranes were seen between subunits derived from free and from membrane-bound ribosomes. (4) It is concluded that the binding of ribosomes to membranes does not require that they be translating a messenger RNA, and that the mechanism whereby bound and free ribosomes synthesize different groups of proteins does not depend on two groups of ribosomes that differ in their ability to bind to endoplasmic reticulum.     

Internal pH can regulate Ca2+ uptake and the acrosome reaction in sea urchin sperm

The egg jelly-induced acrosome reaction of sea urchin sperm is accompanied by intracellular alkalinization and Ca2+ entry. We have previously shown that in the absence of egg jelly, NH4Cl, which increases intracellular pH (pHi), induces Ca2+ uptake and the acrosome reaction in sperm of the sea urchin, Strongylocentrotus purpuratus. Here we show that at a constant concentration of NH4Cl (20 mM) in seawater, sperm react less as external pH is lowered from the normal 8 to 7.25. The pH dependence of the NH4Cl response is not very sensitive to temperatures between 12 and 17 degrees C. NH4Cl (15-50 mM) stimulates Ca2+ uptake and acrosome reactions in sperm suspended in Na+-free seawater, a condition known to inhibit the inductive effect of jelly. Jelly does not further stimulate Ca2+ uptake of sperm preincubated in NH4Cl, indicating that once the permeability to Ca2+ is increased by raising the pHi, the jelly has no further effect. We have used the membrane potential-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide to follow the membrane potential change that occurs when NH4Cl is added. Depolarization (25 mV) is associated with the acrosome reaction when either the natural inducer, egg jelly, or NH4Cl is added to sperm. Response to both inducers is inhibited under conditions known to abolish the acrosome reaction, i.e., low-pH seawater and nisoldipine. These results indicate that the NH4Cl-induced depolarization that accompanies the reaction is probably due to the opening of channels that allow Ca2+ to enter the cell and not to the depolarization by NH4+ ions. High-K+ seawater, which depolarizes sperm, and tetraethylammonium, a K+ channel blocker, inhibit the jelly-induced depolarization and the acrosome reaction, but do not inhibit NH4Cl-induced changes. It has already been shown that nigericin promotes Ca2+ entry and the acrosome reaction in sea urchin sperm. We found that the action of this ionophore depends on the pH of normal seawater. In the absence of external Na+ (replaced by choline), nigericin does not induce the reaction and does not stimulate Ca2+ uptake.     

A novel method for the rapid quantitative determination of ribosome dissociation factor (IF-3) activity. An assay based on the coupling of the dissociation and peptidyltransferase reactions

An assay for factor-dependent ribosome dissociation has been developed, by coupling with the peptidyltransferase reaction between acylaminoacyl-tRNAs and puromycin. The peptidyltransferase reaction is specific for 60S subunits; it is inhibited by derived 40S subunits which combine with derived 60S subunits to form ribosomes. Native 40S subunits, and derived 40S subunits treated with an extract (IF-3) obtained from native 40S subunits, do not interact with 60S subunits and they do not affect the 60S-dependent peptidyltransferase reaction. The native 40S extract releases subunits from 80S ribosomes and the 60S subunits released, a measure of dissociation, can be determined by the peptidyltransferase reaction.     

Action of methanol on the assocation of ribosomal subunits and its effect on the GTPase activity of elongation factor G

Methanol causes association of 30S and 50S ribosomal subunits from $\text{E. coli}$ at MgCl₂ concentrations in which they are normally completely dissociated. The 70S ribosome formed under these conditions shows a lower sedimentation velocity and is functionally active in the EF-G GTPase. Association of ribosomal subunits in the presence as well as absence of methanol is affected by washing the ribosomes with 0.5 M NH₄Cl. Methanol reduces the Mg²⁺ concentration required for subunit association as well as for EF-G GTPase activity. The basic requirement for EF-G GTPase activity both with and without alcohol is shown to be the association of 30S and 50S subunits.     

Ribosomal complexes from an extremely halophilic bacterium and the role of cations

Concentrated extracts of Halobacterium cutirubrum were prepared at 0 C by gently disrupting cells with a nonionic detergent in a medium containing 3.0 m KCl, 0.5 m NH(4)Cl, and 0.04 m (or more) magnesium acetate and then treating the gelatinous mass with deoxyribonuclease. On KCl-sucrose gradients containing 0.5 m NH(4)Cl and 0.04 m magnesium acetate, these extracts showed 30S and 50S ribosomal subunits plus a flat profile of faster-sedimenting material up to high S values. Only after frozen storage or brief incubation of the extract were 70S ribosomes and distinct classes of small polyribosomes detected. Digestion with ribonuclease converted faster-sedimenting material to 70S particles. The presence of chloramphenicol during preparation of the extracts did not affect these results. The evidence suggests that ribosomal particles exist in these cells as subunits or as polyribosomes but not as 70S ribosomes. To investigate the function of Mg(++) and NH(4) (+) ions in ribosomal complexes from this halophile, concentrated cell extracts and extracts incubated with (14)C-leucine were examined on KCl-sucrose gradients containing different concentrations of these ions. Polyribosomes and the bulk of 70S ribosomes dissociated reversibly to subunits at about 0.01 m Mg(++), whereas a small fraction of the 70S particles, including those which in vitro incorporated (14)C-leucine into nascent protein, dissociated only below 1 mm Mg(++). Below this concentration of Mg(++), nascent protein remained attached to the 50S subunit even at 0.04 mm Mg(++) in the presence of 0.35 to 0.5 m NH(4)Cl. Nascent protein, presumably as peptidyl-transfer ribonucleic acid, dissociated reversibly from 50S subunits only at 0.04 mm Mg(++) and 0.1 m or less NH(4) (+). Thus, the stability of polyribosomes from H. cutirubrum depends specifically on both Mg(++) and NH(4) (+) ions.