Total reconstitution of active small ribosomal subunits of the extreme halophilic archaeon Haloferax mediterranei

The small ribosomal subunit of the halophilic archaeon Haloferax mediterranei has been reconstituted from its dissociated rRNA and protein components. Efficient reconstitution of particles, fully active in poly(U)-dependent polyphenylalanine synthesis, occurs after 2 h of incubation at 36°C in the presence of l.5 M of (NH₄)₂SO₄ 100 mM of MgAc₂, 20 mM Tris-HCI (pH 8.2) and 6 mM 2-mercaptoethanol. Important differences in the optimal ionic conditions for the reconstitution of the 30S and the 50S ribosomal subunits from Haloferax mediterranei have been found. K⁺ and NH₄⁺ ions have differing abilities to promote the reconstitution of the particles. The assembly of 30S ribosomal subunits of H. mediterranei has a higher tolerance to ionic strength than the assembly of the 50S subunits and it is independent of the Mg²⁺concentration present in the system.     

Loosening and unfolding of E. coli 50 S ribosomal subunits: Dependence on magnesium content and temperature

Reversible change of 50 S ribosomal subunits to 40 S particles takes place in cold buffered 0.5 M NH₄Cl solutions either containing Mg⁺⁺ (up to 0.1 M), or free from Mg⁺⁺ and even supplemented with EDTA (1 mM). The 40 S particles were stable only within a definite temperature range. Heating of the samples caused completely irreversible unflding of the 40 S particles. This melting appeared to be co-operative and took place within a very narrow range of temperature, which for samples containing Mg⁺⁺ was a linear function of the log of Mg⁺⁺ concentration.The results suggest that two types of bonds maintained the compact structure of the ribosomal subunits: ionic bonds involving Mg⁺⁺ and heat-labile weak interactions between ribosomal components.     

Mitochondrial ribosomal RNA genes of yeast: Their mutations and a common nuclear suppressor

Summary Due to the absence of repetition of the rRNA genes in S. cerevisiae mitochondria, isolation of ribosomal mutants at the level of the rRNA genes is relatively easy in this system. We describe here a novel thermosensitive mutation, ts1297, localized by rho^- deletion mapping in (or very close to) the sequence corresponding to the small ribosomal RNA (15S) gene. Defective mutations of the small rRNA have not been reported so far.In the mutant, the amount of 15S rRNA and of the small ribosomal subunit, 37S, is reduced. The quantity of the large ribosomal RNA (21S), directly extracted from mitochondria, appears normal. However, the large ribosomal subunit, 50S, seems to be fragile and could be recovered only in the presence of Ca²⁺ in place of Mg²⁺. The 50S particles seem to be completely degraded under normal conditions of extraction with Mg²⁺.The thermosensitive phenotype of the ts1297 mutant is suppressed by a nuclear mutation SU₁₀₁. The SU₁₀₁ mutation had been originally isolated as a suppressor of another mitochondrial mutation, ts902, which is located within the 21S rRNA gene.These results suggest that the mitochondrial mutations ts1297 and ts902 are both involved in the interaction of the large and small ribosomal subunits.     

Effective formaldehyde fixation of the reversibly interacting ribosomal system

The possiblity of using formaldehyde fixation for the quantitative studies of the reversibly interacting ribosomal system 50S–30S 50S+30S was investigated. Conditions have been found under which formaldehyde fixation of a reaction mixture of purified ribosomal particles proved to be possible without any change in the initial ratio of its components. This fixation technique was used in studying the dependence of the association level on Mg⁺⁺ ion concentration. An estimation has been made of the number of Mg⁺⁺ ions which bind during the association of 50S and 30S subparticles.     

Kinetics of the reversible association of ribosomal subunits: Stopped-flow studies of the rate law and of the effect of Mg2+

The kinetics of association and dissociation of Escherichia coli 30 S and 50 S ribosomal subunits appear to fit the simple scheme

over a wide range of Mg²⁺ and ribosome concentrations, for the preparations studied (which have a sharp [Mg²⁺]-dependence on the equilibrium degree of association, e.g. 10% to 90% for 1.5 mm to 3.5 mm). Both rate constants depend strongly upon magnesium ion concentration (k₂ goes from 0.04 × 10⁶ to 21 × 10⁶m⁻¹ s⁻¹ as [Mg²⁺] goes from 1.5 mm to 8 mm; k₁ goes from 150 to 2 s⁻¹ in the interval 1.0 to 3.0 mm), but k₁ may level off above 3.0 mm and k₂ increases slowly at high [Mg²⁺]. (The highest rate may not be far from the diffusion-controlled limit.) The primary effect of Mg²⁺, as calculated from the rather large changes in binding as a function of [Mg²⁺], is to decrease the contribution of electrostatic repulsion to the free energy of activation; specific, or class-specific, interactions of di- and multivalent cations with unknown ribosomal substructures may modulate this effect.     

Association of nascent polypeptide with 30S ribosomal subunits

1. Crude extracts of Escherichia coli were used to synthesize nascent peptides under the direction of endogenous mRNA and in the presence of radioactive amino acids. Analysis of such extracts by sucrose-gradient centrifugation in low Mg²⁺ concentration has shown that after 2min of incubation approximately 14% of the total labelled protein recovered on the gradient, in association with whole ribosomes, sediments with 30S ribosomal subunits; this value rises to approximately 24% after 30min of incubation. The labelled protein associated with 30S ribosomal subunits is insoluble in hot trichloroacetic acid. 2. Similar results were also obtained in extracts that synthesized polypeptides under the direction of either of the synthetic polyribonucleotides poly(A) or poly(A,G,C,U). In contrast, however, analysis of crude extracts programmed in protein synthesis by poly(U) has indicated that under these conditions 30S ribosomal subunits have no associated polyphenylalanine; similarly there is little associated peptide after programming of extracts by poly(U,C).     

Purification of 30S ribosomal subunit by streptavidin affinity chromatography

Preparation of pure ribosomal subunits carrying lethal mutations is necessary for studying every essential functional region of ribosomal RNA. Affinity purification via a tag, inserted into rRNA proved to be procedure of choice for purification of such ribosomal subunits. Here we describe fast and simple purification method for the 30S ribosomal subunits using affinity chromatography. Streptavidin-binding tag was inserted into functionally neutral helix 33a of the 16 S rRNA from Escherichia coli. Tagged ribosomal subunits were shown to be expressed in E. coli and could be purified. Purified subunits with affinity tag behave similarly to the wild type subunits in association with the 50S subunits, toe-printing and tRNA binding assays. Tagged 30S subunits could support cell growth in the strain lacking wild type 30S subunits and only marginally change the growth rate of bacteria. The presented purification method is thus suitable for further use in purification of 30S subunits carrying any lethal mutations.     

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.     

Ribosome crystallization in chicken embryos. I. Isolation, characterization, and in vitro activity of ribosome tetramers

Isolated tetrameric particles (166S) derived from the crystalline lattices known to appear in hypothermic chicken embryos consist of mature 80S ribosomes which contain all species of ribosomal RNA and a complete set of ribosomal proteins. Ribosome tetramers are not a special type of polysomes since in solutions of high ionic strengths (500 mM KCl and 50 nM triethanolamine-HCl buffer) containing 5 mM MgCl₂ they dissociate into 40S and 60S ribosomal subunits, without the need of puromycin, and at a concentration of Mg⁺⁺ higher than 3 mM they are not disassembled by mild RNase treatment. Tetramers spontaneously disassemble into 80S monomers when the Mg⁺⁺ concentration is lowered to 1 mM at relatively low ionic strength. Tetramers failed to couple in vitro puromycin-³H into an acid-insoluble product, indicating the lack of nascent polypeptide chains. Although tetramers have no endogenous messenger RNA activity, they can be programmed in vitro with polyuridylic acid (poly U) to synthesize polyphenylalanine. All ribosomes within a tetramer can accept poly U, without the need of disassembly of the tetramers into monomers or subunits.     

Viomycin favours the formation of 70S ribosome couples

Summary The peptide antibiotic viomycin at a concentration of 10 M inhibits E. coli ribosomes to the extent of about 70% as measured in the poly(U) system, and to about 85% in a natural mRNA (R17) system. Ribosomes from M. smegmatis show no activity at all at this concentration of the antibiotic. Experiments on the Mg²⁺ dependent dissociation and association of the ribosomal subunits revealed that viomycin stabilizes the 70S couples and promotes association of ribosomal subunits. This response is related to the drug action as indicated by the observation that viomycin resistant strains are not affected by viomycin with respect to dissociation and 70S couple information. A model for the inhibitory action of the drug is proposed.     

Stabilization of 30 S ribosomal subunits of Bacillus subtilis W168 by spermidine and magnesium ions

An explanation for the fragility of 30 S ribosomal subunits of Bacillus subtilis has been studied. Degradation of 16 S ribosomal RNA, rather than degradation of ribosomal proteins, was found to cause the inactivation of 30 S subunits. Although RNAases were bound specifically to 30 S ribosomal subunits, the RNAases were able to function. Spermidine was found to contribute to the stabilization of 30 S ribosomal subunits by inhibiting the degradation of 16 S ribosomal RNA. A high concentration of Mg²⁺ also stabilized the 30 S ribosomal subunits of Bacillus subtilis. The polypeptide synthetic activity of 30 S ribosomal subunits prepared in the presence of spermidine was at least 4-times greater than that of 30 S ribosomal subunits prepared in the absence of spermidine; this activity was maintained without any loss for 3 months at ?70°C.     

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 of the second tetracycline binding site on the ribosomal small subunit of E.coli

Tetracycline blocks stable binding of aminoacyl-tRNA to the bacterial ribosomal A-site. Various tetracycline binding sites have been identified in crystals of the 30S ribosomal small subunit of Thermus thermophilus. Here we describe a direct photo- affinity modification of the ribosomal small subunits of Escherichia coli with 7-[³H]-tetracycline. To select for specific interactions, an excess of the 30S subunits over tetracycline has been used. Primer extension analysis of the 16S rRNA revealed two sites of the modifications: C936 and C948. Considering available data on tetracycline interactions with the prokaryotic 30S subunits, including the presented data (E.coli), X-ray data (T.thermophilus) and genetic data (Helicobacter pylori, E.coli), a second high affinity tetracycline binding site is proposed within the 3′-major domain of the 16S rRNA, in addition to the A-site related tetracycline binding site.     

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.     

A cold-sensitive cytoplasmic mutation of Saccharomyces cerevisiae affecting assembly of the mitochondrial 50S ribosomal subunit.

Summary A cytoplasmic mutant of Saccharomyces cerevisiae (E23-1) has been isolated that is resistant to erythromycin and cold sensitive for growth on nonfermentable carbon sources at 18°. Genetic analysis has shown that both of these properties probably result from a single mutation at the rib2 locus which maps close to or within the gene for the 21S rRNA of the mitochondrial 50S ribosomal subunit. Electrophoresis of total RNA extracted from purified mitochondria demonstrated that the 21S and 14S rRNA species from both mutant and wild-type cells were present in roughly equimolar quantities regardless of growth temperature. The mutant is therefore not defective in the synthesis of the 21S rRNA. Sucrose gradient analysis of the mitochondrial ribosomes in Mg²⁺-containing buffers revealed that approximate values for the ratio of 50S to 37S subunits were 1:1 for wild-type cells grown at either 18° or 32°, 0.5:1 for the mutant grown at 32° and 0.2:1 for the mutant grown at 18°. The subunit ratios were approximately 1:1 when Ca²⁺-containing buffers were used, however, In alls cases, 50S particles from the mutant grown at 18° lacked or contained markedly reduced amounts of two distinctive protein components that were present in the mutant at 32° and in the wild-type at both temperatures. In addition, no intact 21S RNA could be recovered from the mitochondrial ribosomes of the mutant grown at the restrictive temperature, even in the presence of Ca²⁺. These findings indicate that mitochondrial 50S ribosomal subunits produced by the mutant at 18° are structurally defective and raise the possibility that the defect results from an alteration in the gene for 21S rRNA.A preliminary report of this work was presented at the meeting on The Molecular Biology of Yeast, Cold Spring Harbor Laboratory, August 18–22, 1977     

A recurrent magnesium-binding motif provides a framework for the ribosomal peptidyl transferase center

The ribosome is an ancient macromolecular machine responsible for the synthesis of all proteins in all living organisms. Here we demonstrate that the ribosomal peptidyl transferase center (PTC) is supported by a framework of magnesium microclusters (Mg²⁺-μc's). Common features of Mg²⁺-μc's include two paired Mg²⁺ ions that are chelated by a common bridging phosphate group in the form Mg_(a)²⁺–(O1P-P-O2P)–Mg_(b)²⁺. This bridging phosphate is part of a 10-membered chelation ring in the form Mg_(a)²⁺–(OP-P-O5′-C5′-C4′-C3′-O3′-P-OP)–Mg_(a)²⁺. The two phosphate groups of this 10-membered ring are contributed by adjacent residues along the RNA backbone. Both Mg²⁺ ions are octahedrally coordinated, but are substantially dehydrated by interactions with additional RNA phosphate groups. The Mg²⁺-μc's in the LSU (large subunit) appear to be highly conserved over evolution, since they are unchanged in bacteria (Thermus thermophilus, PDB entry 2J01) and archaea (Haloarcula marismortui, PDB entry 1JJ2). The 2D elements of the 23S rRNA that are linked by Mg²⁺-μc's are conserved between the rRNAs of bacteria, archaea and eukarya and in mitochondrial rRNA, and in a proposed minimal 23S-rRNA. We observe Mg²⁺-μc's in other rRNAs including the bacterial 16S rRNA, and the P4–P6 domain of the tetrahymena Group I intron ribozyme. It appears that Mg²⁺-μc's are a primeval motif, with pivotal roles in RNA folding, function and evolution.     

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.     

Stimulation by polyamines of enzymatic methylation of two adjacent adenines near the 3′ end of 16S ribosomal RNA of Escherichia coli

Effect of polyamines on the methylation of adenine in 16S rRNA was examined using the purified methylating enzyme. When 23S core particles were used as substrate, the activity was stimulated by Mg²⁺, Ca²⁺ and monovalent cations. Even in the presence of optimal concentrations of Mg²⁺ and NH₄⁺, the addition of 1 mM spermidine stimulated the methylation approximately 1.7-fold. When 30S ribosomal subunits were used as substrate, the rate of methylation was 20% of that of the methylation of 23S core particles. The activity was not influenced significantly by Mg²⁺, Ca²⁺ or monovalent cations. The addition of spermidine inhibited the methylation.