Reversible modification of Escherichia coli ribosomes with 2,3-dimethylmaleic anhydride. A new method to obtain protein-deficient ribosomal particles.

Treatment of Escherichia coli ribosomes with the protein reagent 2,3-dimethylmaleic anhydride is accompanied by inactivation of polypeptide polymerization and by dissociation of ribosomal proteins. Regeneration of the modified amino groups at pH 6.0 is followed by reactivation and reconstitution of the ribosomes. Prior to regeneration of the amino groups, ribosomal particles and split proteins can be separated by centrifugation, which allows the preparation of new protein-deficient particles. The ribosomal particles obtained by three successive treatments with 2,3-dimethyl-maleic anhydride at a molar ratio of reagent to ribosome equal to 16,000 lack proteins S1, S2, S3, S5, S10, S13, S14, L7, L8, L10, L11, L12, and L20 and have lost part of proteins S4, L1, L6, L16, and L25. This new procedure to obtain protein-deficient ribosomal particles is mild and might be useful to dissociate other protein-containing structures in addition to ribosomes.     

Identification of proteins involved in the peptidyl transferase activity of ribosomes by chemical modification.

Photochemical oxidation of Escherichia coli 50 S ribosomal subunits in the presence of methylene blue or Rose Bengal causes rapid loss of peptidyl transferase activity. Reconstitution experiments using mixtures of components from modified and unmodified ribosomes reveal that both RNA and proteins are affected, and that among the proteins responsible for inactivation there are both LiCl-split and core proteins. The proteins L2 and L16 from the split fraction and L4 from the core fraction of unmodified ribosomes were together nearly as effective as total unmodified proteins in restoring peptidyl transferase activity to reconstituted ribosomes when added with proteins from modified ribosomes. These three proteins are therefore the most important targets identified as responsible for loss of peptidyl transferase activity on photo-oxidation of 50 S ribosomal subunits.     

Additional Nucleotide Sequences in Precursor 16S Ribosomal RNA from <Emphasis Type="Italic">Escherichia coli</Emphasis>

MATURE 5S, 16S and 23S ribosomal RNA species present in E. coli ribosomes are the end products of complex biosyn-thetic pathways. They are formed by reduction in length, and methylation of longer RNA chains transcribed on the ribosomal RNA cistrons of E. coli DNA. While these modifications take place the ribosome structure is formed by progressive addition of ribosomal proteins and conformational changes in the resulting ribonucleoprotein precursor particles¹.     

The isolation and properties of cardiac ribosomes and polysomes

1. A method is described by which good yields of ribosomes and polysomes free of contamination by submitochondrial fragments can be prepared from rat cardiac muscle. These preparations are capable of incorporation of amino acids into protein in vitro. 2. The ribosome preparation consists of 32% of monomeric ribosomes and 68% of ribosomal aggregates or polysomes. The polysome preparation has a decreased monomeric content. Dimers, trimers, tetramers, pentamers and larger components can be differentiated. 3. The polysome aggregate structure is degraded to monomeric ribosomes on incubation with small amounts of ribonuclease or by preparation in the absence of Mg²⁺ ions. The degradation in the absence of Mg²⁺ ions was not reversible and drastically decreased the incorporation of amino acids in vitro. 4. The cardiac ribosomes contained two major RNA species sedimenting at 19s and 28s in a 1:2·4 ratio. 5. The RNA/protein ratio of cardiac ribosomes and polysomes was consistently lower than that of similar preparations from liver. The concentrations of Na⁺ and K⁺ ions present during preparation had a great effect on the RNA/protein ratio. 6. Optimum conditions for the incorporation of amino acids into protein in vitro are reported. Cardiac ribosomes have a lower rate of incorporation of amino acids in vitro than liver ribosomes. 7. Heart cell sap is less active than liver cell sap: evidence is presented that a factor, present in liver cell sap and concerned with stimulating the synthesis of the peptide chain, is lacking in heart cell sap. 8. Pulse-labelling of perfused hearts followed by examination of the subcellular structures showed that the ribosomal fraction was the most active in the incorporation of amino acids in vitro.     

RIBONUCLEOPROTEIN PARTICLES IN THE AMPHIBIAN OOCYTE NUCLEUS : Possible Intermediates in Ribosome Synthesis

Studies of the sedimentation properties of RNP¹ material from the nucleus of the amphibian oocyte have indicated (1) that there are few, if any, 78S ribosomes in the nucleus, (2) that there are smaller particles sedimenting at 50-55S and 30S, and (3) that the larger of these is the precursor of the 60S subunit of the cytoplasmic ribosomes. Although the nature of the 30S material is not completely clear, it probably includes precursor particles to the 40S ribosomal subunit. Heavy (50-55S) particles are predominant in immature oocytes of Triturus viridescens, whereas in immature oocytes of Triturus and Amblystoma mexicanum they are reduced greatly in amount, but are still detectable. Double-labeling studies of RNA and protein reveal that both types of particle incorporate uridine-³H, but that the 50-55S material of immature oocytes does not incorporate ¹⁴C-labeled amino acids. However, other evidence exists that favors the RNP nature of this material. Sedimentation analyses after SDS extraction show that 50-55S particles contain 40 and 30S RNA, whereas 30S particles contain 20S RNA. These types of RNA represent at least 80% of all the extractable nuclear RNA. The 50-55S particles are probably heterogeneous, including both particles containing mostly 40S RNA and particles containing only 30S RNA.     

Structure and function of 5S RNA: the role of the 3'' terminus in 5S RNA function

Summary 5S RNA from B. stearothermophilus and E. coli was reacted with NaIO₄ and aniline to remove their 3 terminal nucleoside. These modified 5S RNA molecules were then incorporated in B. stearothermophilus 50 S ribosomal subunits and tested for biological activities. 50 S ribosomes containing the modified 5S RNAs exhibited full activity and we therefore conclude, that the 3 terminus of 5S RNA does not play an active role in protein synthesis.     

RNA Synthesis and polysome formation in pollen tubes

The formation of polysomes in relation to RNA synthesis was investigated in tobacco (Nicotiana tabacum L.) pollen cultivated submersely for a period of 12 h. The percentage of polysomes was estimated by determining the number of ribosomes carrying nascent polypeptides using RNase and 0.8 M KC1 treatment of the ribosomal preparation. This approach showed that the proportion of ribosomes “active” in protein synthesis amounts to about 12 % in dry pollen rising to 46 % within 10 min of imbibition and to 66 % during the period 1–4 h of cultivation. The latter increase is accompanied by a rapid incorporation of uridine-¹⁴C into polysome-as-sociated RNA and is sensitive to actinomycin D. The rapidly labelled RNA isolated from the ribosomal preparation sedirnented in the range from about 5S to 30S. Longer labelling periods led to a gradual shift of the major peak of this heterogeneous RNA from about 11.5S and 14S to about 7.5S and the development of radioactivity peaks in the position of 18S and 28S RNA. Uridine incorporated both into the active ribosomes and into the subunits of inactive ribosomes at a more or less constant rate during the whole 12-h period of cultivation. These results present evidence that in addition to the initial combination of the existing ribosomes with the stored mRNA following imbibition, an activation of pollen tube genome and its implication in directing protein synthesis take place during the early phases of pollen tube growth. From the results on kinetics of labelling of ribosomes it appears that in pollen tubes new synthesized ribosomal subunits enter polysomes directly, the entry of 40S subunits being more rapid than that of 60S subunits.     

Functional Inactivation and Appearance of Breaks in RNA Chains Caused by Gamma Irradiation of Escherichia coli Ribosomes

70S ribosomes and 30S ribosomal subunits from Escherichia coli MRE 600 were exposed to gamma irradiation at -80szC. Exponential decline of activity with dose was observed when the ability of ribosomes to support the synthesis of polyphenylalanine was assayed. Irradiated ribosomes showed also an increased thermal lability. D₃₇ values of 2.2 MR and 4.8 MR, corresponding to radiation-sensitive molecular weights of 3.1 × 10⁵ and 1.4 × 10⁵, were determined for inactivation of 70S ribosomes and 30S subunits, respectively. Zone sedimentation analysis of RNA isolated from irradiated bacteria or 30S ribosomal subunits showed that at average, one chain scission occurs per four hits into ribosomal RNA. From these results it was concluded that the integrity of only a part of ribosomal proteins (the sum of their molecular weights not exceeding 1.4 × 10⁵) could be essential for the function of the 30S subunit in the polymerization of phenylalanine. This amount is smaller if the breaks in the RNA chain inactivate the ribosome.     

Biochemical research on oogenesis. Comparison between the proteins of the ribosomes and the proteins of the 42 S particles from small oocytes of Xenopus laevis

Previtellogenic oocytes of Xenopus laevis synthesize large amounts of 5 S RNA and transfer RNA, but very little, if any, 28 S and 18 S RNA. About half of the RNA of these oocytes is stored in nucleoprotein particles sedimenting at 42 S. These particles contain 5 S RNA, transfer RNA, and several proteins, the function of which remains so far unknown.The proteins of the 42 S particles were analyzed by two-dimensional electrophoresis on polyacrylamide gel. The resulting fingerprints displayed one major and two minor basic spots. None of these coincided with any of the 37 spots produced by the 60 S subunit of the ribosomes and with the 30 spots produced by the 40 S subunit. We conclude that no ribosomal component other than 5 S RNA is present in the 42 S particles.The fingerprints of 40 S and 60 S ribosomal proteins from X. laevis coincided almost completely with the corresponding fingerprints from the rat and the rabbit.     

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.     

Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly.     

Subunit interactions of rabbit muscle aldolase. Conformational change of aldolase in magnesium chloride and its relationship to the dissociation of subunits.

The effect of Escherichia coli ribosomal protein S1 on translation has been studied in S1-depleted systems programmed with poly(U), poly(A) and MS2 RNA³. The translation of the phage RNA depends strictly on the presence of S1. Optimum poly(U)-directed polyphenylalanine synthesis and poly(A)-programmed polylysine synthesis also require S1. Excess S1 relative to ribosomes and messenger RNA results in inhibition of translation of MS2 RNA and poly(U), but not of poly (A). In the case of phage RNA translation, this inhibition can be counteracted by increasing the amount of messenger RNA. Three other 30 S ribosomal proteins (S3, S14 and S21) are also shown to inhibit MS2 RNA translation. The effects of S1 on poly(U) translation were studied in detail and shown to be very complex. The concentration of Mg²⁺ in the assay mixtures and the ratio of S1 relative to ribosomes and poly(U) are crucial factors determining the response of this translational system towards the addition of S1. The results of this study are discussed in relation to recent developments concerning the function of this protein.     

Messenger ribonucleic acid of cerebral nuclei

1. RNA was isolated from crude nuclear preparations and from ribosomes derived from rat brain and liver. Nuclear RNA was obtained by lysis of the nuclei with sodium dodecyl sulphate, followed by denaturation and removal of DNA and protein with hot phenol. 2. Base composition analyses indicated that the cerebral nuclear RNA preparation contained a higher proportion of non-ribosomal RNA than the analogous hepatic preparation. 3. Sucrose-density-gradient analyses revealed a heterogeneous profile for each nuclear RNA preparation, with two major peaks possessing the sedimentation properties of ribosomal RNA (18s and 28s). 4. Template activities of both preparations were widely distributed through the sucrose density gradients. 5. The cerebral nuclear RNA preparation was more active than the hepatic nuclear RNA preparation in promoting amino acid incorporation in cell-free systems from Escherichia coli and rat brain. 6. Cerebral nuclear RNA stimulated amino acid incorporation in a cerebral ribosomal system even in the presence of an excess of purified E. coli transfer RNA. 7. It is concluded that a significant proportion of cerebral nuclear RNA has the characteristics of messenger RNA.     

Thermal Analysis of Bacterial Ribosomes

Ribosomal particles of E. coli were examined by using a heat leakage scanning calorimeter. Remarkable changes were observed in thermograms of 70S ribosomes and their subunits when the Mg²⁺ concentration was raised from 1 mm to 10 mm. It was suggested that ribosomal subunits exist in more than one conformation, and changes in their conformation might be the primary cause of the association-dissociation process of ribosomes. Comparisons of thermograms of RNase- and chymotrypsin-treated, as well as non-treated SOS and 30S subunits suggest that conformational changes in each subunit may be ascribed to changes in rRNA.     

Reversible modification of 50S ribosomal subunits with dimethylmaleic anhydride

Summary The reversible modification of protein amino groups with dimethylmaleic anhydride, which had already been used to dissociate proteins from the 70S ribosomes of Escherichia coli (Pintor-Toro, J. A., et al. (1979) Biochemistry 18, 3219) was applied to the preparation of protein-deficient particles from the 50S subunits. Three successive cycles of treatment with dimethylmaleic anhydride, separation of dissociated proteins and regeneration of the modified amino groups produce partially inactivated ribosomal cores lacking proteins L7, L11 and L12, and having very small amounts of L1, L6 and L10. Incubation of these cores with the corresponding split proteins is accompanied by complete reactivation of the polypeptide synthesizing activity as compared with control 50S subunits.Abbreviation DMMA 2,3-dimethylmaleic anhydride     

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.     

The Kinetics of the Synthesis of Ribosomal RNA in E. coli

The kinetics of the synthesis of ribosomal RNA in E. coli has been studied using C¹⁴-uracil as tracer. Two fractions of RNA having sedimentation constants between 4 and 8S have kinetic behavior consistent with roles of precursors. The first consists of a very small proportion of the RNA found in the 100,000 g supernatant after ribosomes have been removed. It has been separated from the soluble RNA present in much larger quantities by chromatography on DEAE-cellulose columns. The size and magnitude of flow through this fraction are consistent with it being precursor to a large part of the ribosomal RNA.

A fraction of ribosomal RNA of similar size is also found in the ribosomes. This fraction is 5 to 10 per cent of the total ribosomal RNA and a much higher proportion of the RNA of the 20S and 30S ribosomes present in the cell extract. The rate of incorporation of label into this fraction and into the main fractions of ribosomal RNA of 18S and 28S suggests that the small molecules are the precursors of the large molecules. Measurements of the rate of labeling of the 20, 30, and 50S ribosomes made at corresponding times indicate that ribosome synthesis occurs by concurrent conversion of small to large molecules of RNA and small to large ribosomes.

     

Structural Transitions in Ribonucleic Acid During Ribosome Development

Amino acid deprivation of a "relaxed" auxotroph of Escherichia coli results in the accumulation of protein-deficient, immature ribosomes ("relaxed particles"). The ribonucleic acid (RNA) of these particles was shown to differ from mature ribosomal RNA in both sedimentation characteristics and in elution from columns of methylated albumin-keiselguhr. When relaxed particles were allowed to become converted to mature ribosomes, the unique properties of the RNA were lost, and this RNA became indistinguishable from mature RNA. The conversion of relaxed particles to ribosomes did not involve degradation and resynthesis of RNA. It is concluded that ribosomal RNA undergoes a configurational transition during ribosome development, and that this transition is not the result of changes in the primary structure of the RNA.     

ABERRANT INTRANUCLEOLAR MATURATION OF RIBOSOMAL PRECURSORS IN THE ABSENCE OF PROTEIN SYNTHESIS

To help elucidate the role of protein in the maturation of ribosomal RNA in cultured L cells, we have studied the effects of cycloheximide upon the maturation process and upon the intranucleolar ribonucleoprotein particles containing the "preribosomal RNA's." Five parameters of these particles were analyzed: (a) extractability, (b) sedimentation characteristics in sucrose gradients, (c) RNA composition, (d) buoyant density in CsCl gradients, and (e) effects of increased ionic strength on the buoyant density. When protein synthesis is inhibited, the rate of conversion of the precursor 45S ribosomal RNA is rapidly diminished, falling to less than 30% of the control rate within 1 hr. Nevertheless, in terms of the first three parameters there is no difference between control and cycloheximide nucleolar particles. However, the cycloheximide particles have a lower and more heterogeneous buoyant density and a more variable response to increased ionic strength. The results imply that the protein composition of the cycloheximide particles is different from that of particles from control cells, and that the entire protein complement is not necessary for the first cleavages in the maturation process, although it is necessary for the normal rate of processing and for the eventual appearance of both 18S and 28S rRNA in mature ribosomes.