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.     

An excess of the small ribosomal subunits and a higher rate of turnover of the 60 S than of the 40 S ribosomal subunits in L cells grown in suspension culture.

A considerable excess of small ribosomal subunits was observed in L cells grown in suspension culture. The ratio between the small and large ribosomal subunits in the cytoplasm was estimated to be 1.17 ± 0.05 for cells dividing every 20 to 24 hours.The 60 S ribosomal subunits were turning over much faster than the 40 S subunits. Half-lives of 155 ± 20 hours for 18 S ribosomal RNA and 82 ± 15 hours for 28 S ribosomal RNA were observed under conditions where the cell number doubled every 24 hours and the viability was 95%. By correcting for cell death the half-lives of 18 S and 28 S ribosomal RNA were estimated to be approximately 300 hours and 110 hours, respectively. During storage of isolated ribosomes the small ribosomal subunits were degraded faster than the large subunits. This shows that the degradation of 60 S subunits was not an artifact taking place during the isolation procedure.It is postulated that the small ribosomal subunits are protected by protein to a greater extent than the 60 S subunits in these rapidly growing cells in suspension culture. The protection may take place both in the nucleus during synthesis, thus avoiding degradation (“wastage”) of nascent subunit precursors, and later in the cytoplasm. A calculation has been carried out to show that the observed excess of small subunits may be accounted for on the basis of a 1:1 synthesis of the small and large ribosomal subunits in the nucleus and different degradation rates in the cytoplasm. The results do not exclude the possibility of a difference in the “wastage” of 18 S and 28 S ribosomal RNA in the nucleus in addition to the difference in the turnover rates in the cytoplasm.     

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.     

Comparison of native and KCl treated 40S ribosomal subunits from the silkmoth Antherea pernyi and mammals

Native 40S ribosomal subunits and 18S ribosomal RNA from ovarian follicles of the silkmoth A. pernyi showed a lower sedimentation coefficient in comparison to ascites cells, in contrast to the KCl treated 40S ribosomal subunits where no difference was observed in both tissues. Moreover the silkmoth native 40S ribosomal subunits--in contrast to the KCl treated ones--could not reassociate with radioactive ascites cell 60S ribosomal subunits. These results, combined with the great similarities in the two dimensional electrophoretic patterns of 40S ribosomal proteins from silkmoth follicles and other mammalian cells lead to the possibility of the existence of a specific RNase associated with the 40S ribosomal subunit.     

Proteolytic degradation of ribosomal proteins during isolation of ribosomes and ribosomal subunits from Tetrahymena

A preparation procedure previously used to isolate active ribosomal subunits from an amicronucleate strain of Tetrahymena of undefined phenoset (T. «pyriformis CGL) yields inactive subunits when applied to other amicronucleate or to micronucleate strains of this protozoa.Proteolytic degradation of a small number of ribosomal proteins during preparation of ribosomal subunits from these strains explains this results. If cell extraction and ribosome isolation are carried out in the presence of iodoacetamide, proteolytic activity is inhibited and active ribosomal subunits are obtained. Comparison of the protein complements of active ribosomal subunits prepared in the presence of iodoacetamide from three amicronucleate strains of Tetrahymena reveals small but significant differences.     

Electron microscopic study of free and ribosome bound rRNAs from E. coli

The morphology of ribosomal subunits, nucleoprotein cores, and free rRNAs from Escherichia coli has been studied by two high resolution electron microscopic techniques. Conventional transmission electron micrographs showed unfolding of 30S and 50S ribosomal subunits with increasing depletion of proteins. With dedicated (0.3 nm resolution) scanning transmission electron microscopy we were able to visualize unstained freeze dried ribosomal subunits and free rRNAs without artifacts of staining and structural distortion by air drying. By this technique, we have also determined the mass of individual ribosomal particles and rRNA molecules. While the ribosomal subunits displayed their characteristic structural features and mass, free rRNAs appeared unfolded and polydisperse. These results provide direct evidence for distinct conformational differences between free rRNAs and native ribosomal subunits of E. coli.     

Studies on the distribution of ribosomal proteins in mammalian ribosomal subunits.

Murine L5178Y cell ribosomes were dissociated into subunits either with potassium chloride in the presence of puromycin or with the chelating agent EDTA. The proteins of ribosomal subunits obtained by these different methods were compared by means of bidimensional polyacrylamide gel electrophoresis. KCl-derived 60S and 40S subunits were shown to contain 38 and 31 proteins respectively, 3 of them having identical electrophoretic mobilities. Preparations of EDTA-dissociated ribosomal subparticles contained different proportions of these proteins, and 11 major spots were shared between the EDTA-derived large and small ribosomal subunits. Furthermore, 10 proteins absent from subunits treated by high concentrations of KCl were reproducibly found in EDTA-treated ribosomal subparticles.     

Effects of detergents on ribosomal precursor subunits of Bacillus megaterium.

Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.     

Factors causing release of ribosomal subunits from isolated nuclei of regenerating rat liver in vitro.

Incubation medium II causes release of ribosomal subunits from isolated prelabeled nuclei of regenerating rat liver in vitro (Sato, T., Ishikawa, K. and Ogato, K. (1976) Biochim. Biophys. Acta 000, 000-000). The effects of individual components of this medium on release of subunits were studied and the following results were obtained. 1. Dialyzed cytosol was effective in causing release of total labeled RNA, but its effect on release of labeled ribosomal subunits was rather lower than that of low molecular yeast RNA. Spermidine inhibited the release of total labeled RNA as well as that of labeled ribosomal subunits. 2. Low molecular yeast RNA was the most effective component for inducing release of labeled ribosomal subunits. Homologous ribosomal RNA was as effective as yeast RNA. Cytoplasmic ribosomes, prepared by washing with solution of high salt concentration, and their subunits were also effective. 3. Transfer RNA was not so effective as yeast RNA and ribosomal RNA and even after heat treatment it had little effect. 4. Among the homopolyribonucleotides tested, polyuridylic acid had a strong effect but polyadenylic acid, polycytidylic acid and polyinosinic acid had no effect. 5. The effects of yeast RNA and polyuridylic acid in causing release of labeled ribosomal subunits were dependent upon their concentrations in the reaction mixture. The characteristics of the factors which cause release of labeled ribosomal subunits in vitro are discussed on the basis of the results.     

Distinct functions of eukaryotic translation initiation factors eIF1A and eIF3 in the formation of the 40 S ribosomal preinitiation complex.

We have used an in vitro translation initiation assay to investigate the requirements for the efficient transfer of Met-tRNAf (as Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA (or an AUG codon) to form the 40 S preinitiation complex. We observed that the 17-kDa initiation factor eIF1A is necessary and sufficient to mediate nearly quantitative transfer of Met-tRNAf to isolated 40 S ribosomal subunits. However, the addition of 60 S ribosomal subunits to the 40 S preinitiation complex formed under these conditions disrupted the 40 S complex resulting in dissociation of Met-tRNAf from the 40 S subunit. When the eIF1A-dependent preinitiation reaction was carried out with 40 S ribosomal subunits that had been preincubated with eIF3, the 40 S preinitiation complex formed included bound eIF3 (40 S.eIF3. Met-tRNAf.eIF2.GTP). In contrast to the complex lacking eIF3, this complex was not disrupted by the addition of 60 S ribosomal subunits. These results suggest that in vivo, both eIF1A and eIF3 are required to form a stable 40 S preinitiation complex, eIF1A catalyzing the transfer of Met-tRNAf.eIF2.GTP to 40 S subunits, and eIF3 stabilizing the resulting complex and preventing its disruption by 60 S ribosomal subunits.     

Immunochemical analysis of the structure of eukaryotic ribosomes

Summary Antibodies were prepared in rabbits and sheep to rat liver ribosomes, ribosomal subunits, and to mixtures of proteins from the particles. The antisera were characterized by quantitative immunoprecipitation, by passive hemagglutination, by immunodiffusion on Ouchterlony plates, and by immunoelectrophoresis. While all the antisera contained antibodies specific for ribosomal proteins, none had precipitating antibodies against ribosomal RNA. Rat liver ribosomal proteins were more immunogenic in sheep than rabbits, and the large ribosomal subunit and its proteins were more immunogenic than those of the 40S subparticle. Antisera specific for one or the other ribosomal subunit could be prepared; thus it is unlikely that there are antigenic determinants common to the proteins of the two subunits. When ribosomes, ribosomal subunits, or mixtures of proteins were used as antigens the sera contained antibodies directed against a large number of the ribosomal proteins.Abbreviations TP total proteins—used to designate mixtures of proteins from ribosomal particles, hence TP80 is a mixtures of all the proteins from 80S ribosomes - TP60 the proteins from 60S subunits - TP40 the proteins from 40S particles     

DnaK-facilitated ribosome assembly in Escherichia coli revisited

Assembly helpers exist for the formation of ribosomal subunits. Such a function has been suggested for the DnaK system of chaperones (DnaK, DnaJ, GrpE). Here we show that 50S and 30S ribosomal subunits from an Escherichia coli dnaK-null mutant (containing a disrupted dnaK gene) grown at 30 degrees C are physically and functionally identical to wild-type ribosomes. Furthermore, ribosomal components derived from mutant 30S and 50S subunits are fully competent for in vitro reconstitution of active ribosomal subunits. On the other hand, the DnaK chaperone system cannot circumvent the necessary heat-dependent activation step for the in vitro reconstitution of fully active 30S ribosomal subunits. It is therefore questionable whether the requirement for DnaK observed during in vivo ribosome assembly above 37 degrees C implicates a direct or indirect role for DnaK in this process.     

Dephosphorylation of 40S ribosomal subunits by phosphoprotein phosphatase activity from rabbit reticulocytes.

Phosphoprotein phosphatase activities which remove phosphoryl groups from ribosomal protein have been partially purified from rabbit reticulocytes by chromatography on DEAE-cellulose. Two major peaks of phosphoprotein phosphatase activity were observed when 40S ribosomal subunits, phosphorylated $\text{in vitro}$ with cyclic AMP-regulated protein kinases and (γ-³²P)ATP, were used as substrate. The phosphatase activity eluting at 0.14 M KCl was characterized further using ribosomal subunits phosphorylated $\text{in situ}$ by incubation of intact reticulocytes with radioactive inorganic phosphate. Phosphate covalently bound to 40S ribosomal subunits and 80S ribosomes was removed by the phosphatase activity. The enzyme was not active with phosphorylated proteins associated with 60S ribosomal subunits.     

Study on mammalian ribosomal protein reactivity in situ. III. Effect of trypsin on 40S and 60S subunits.

Rat liver 40S and 60S ribosomal subunits were treated with increasing concentrations of trypsin. The activity of both trypsin-treated subunits, when assayed for polyphenylalanine synthesis, progressively decreased, but the 60S subunits were inactivated at much lower trypsin concentrations than were the 40S ones. The sedimentation coefficients of trypsin-treated subunits were identical to those of control subunits when sucrose gradients containing 0.5 M KCl were used. When the sucrose gradients were prepared with a low salt buffer (80 mM KCl), dimer formation was observed with control subunits, but not with trypsin-treated ones. Two-dimensional gel electrophoresis analysis of the proteins extracted from trypsin-treated subunits revealed that all ribosomal proteins in the subunits were accessible to the enzyme. However, several proteins were more resistant to trypsin in compact subunits than when they were free or in unfolded subunits. Proteins of the 60S subunits were generally digested by lower trypsin concentrations than those of the 40S subunits. From the quantitative measurements of the undigested proteins, a classification of the proteins from both subunits according to their trypsin sensitivity was established. These results were compared with those previously obtained concerning ribosomal protein reactivity to chemical reagents.     

Disproportionate accumulation of 18S and 28S ribosomal RNA in cultured normal rat kidney cells treated with picolinic acid or 5-methylnicotinamide

Normal rat kidney cells treated with the pyridine derivative picolinic acid, or 5-methylnicotinamide, an inhibitor of ADP-ribosylation, are unable to process 28S rRNA and accumulate 60S ribosomal subunits in the cytoplasm. Synthesis of polyA(+) RNA, rRNA precursors, and the processing of 18S rRNA into 40S ribosomal subunits are almost unaffected. Serum starvation and treatment of cells with histidinol, cycloleucine, nicotinic acid, or 1,10-phenanthroline do not elicit this alteration in rRNA metabolism. Ribosomal subunits synthesized before picolinic acid addition have different stabilities after picolinic acid treatment. The 40S subunits are degraded while the 60S subunits are more stable, demonstrating that a compensatory mechanism exists to maintain preferentially existing subunits when they are no longer being synthesized. The results suggest that ADP-ribosylation is necessary for proper processing of 28S rRNA and therefore for formation of mature 60S ribosomal subunits.     

Assembly of Membrane-bound Polyribosomes

The 60S large ribosomal subunit binds directly to membranes of the endoplasmic reticulum. Experiments with myeloma cells in tissue culture suggest that membrane-bound polyribosomes are assembled by attachment of small ribosomal subunits and mRNA to membrane-bound 60S subunits.     

The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the ribosome, has been traced to the ArmA/RmtA family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosyl-L-methionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA of the 30S ribosomal subunit. ArmA methylates mature 30S subunits but not 16S rRNA, 50S, or 70S ribosomal subunits or isolated Helix 44 of the 30S subunit. To more fully characterize this family of enzymes, we have investigated the substrate requirements of ArmA and to a lesser extent its ortholog RmtA. We determined the Mg+2 dependence of ArmA activity toward the 30S ribosomal subunits and found that the enzyme recognizes both low Mg+2 (translationally inactive) and high Mg+2 (translationally active) forms of this substrate. We tested the effects of LiCl pretreatment of the 30S subunits, initiation factor 3 (IF3), and gentamicin/kasugamycin resistance methyltransferase (KsgA) on ArmA activity and determined whether in vivo derived pre-30S ribosomal subunits are ArmA methylation substrates. ArmA failed to methylate the 30S subunits generated from LiCl washes above 0.75 M, despite the apparent retention of ribosomal proteins and a fully mature 16S rRNA. From our experiments, we conclude that ArmA is most active toward the 30S ribosomal subunits that are at or very near full maturity, but that it can also recognize more than one form of the 30S subunit.     

Release and recycling of eukaryotic initiation factor 2 in the formation of an 80 S ribosomal polypeptide chain initiation complex.

The eukaryotic initiation factor (eIF)-5 mediates hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. The eIF-2.GDP formed under these conditions is released from the 40 S ribosomal subunit while initiator Met-tRNA(f) remains bound. The released eIF-2.GDP can participate in an eIF-2B-catalyzed GDP/GTP exchange reaction to reform the Met-tRNA(f).eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were also present in an eIF-5-catalyzed reaction, the eIF-2.GDP produced remained bound to the 60 S ribosomal subunit of the 80 S initiation complex. When such an 80 S initiation complex, containing bound eIF-2.GDP, was incubated with GTP and eIF-2B, GDP was released. However, eIF-2 still remained bound to the ribosomes and was unable to form a Met-tRNA(f)l.eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were preincubated with either free eIF-2 or with eIF-2.eIF-2B complex and then added to a reaction containing both the 40 S initiation complex and eIF-5, the eIF-2.GDP produced did not bind to the 60 S ribosomal subunits but was released from the ribosomes. Thus, the 80 S initiation complex formed under these conditions did not contain bound eIF-2.GDP. Under similar experimental conditions, preincubation of 60 S ribosomal subunits with purified eIF-2B (free of eIF-2) failed to cause release of eIF-2.GDP from the ribosomal initiation complex. These results suggest that 60 S ribosome-bound eIF-2.GDP does not act as a direct substrate for eIF-2B-mediated release of eIF-2 from ribosomes. Rather, the affinity of 60 S ribosomal subunits for either eIF-2, or the eIF-2 moiety of the eIF-2.eIF-2B complex, prevents association of 60 S ribosomal subunits with eIF-2.GDP formed in the initiation reaction. This ensures release of eIF-2 from ribosomes following hydrolysis of GTP bound to the 40 S initiation complex.