Properties of ribosomes and RNA synthesized by Escherichia coli grown in the presence of ethionine. V. Methylation dependence on the assembly of E. coli 50 S ribosomal subunits.

An ethionine-containing submethylated particle related to the 50 S ribosomal subunit has been isolated from Escherichia coli grown in the presence of ethionine. This particle (E-50S) lacks L16, contains reduced amounts of L6, L27, L28 and L30 and possesses a more labile and flexible structure than the normal 50 S subunit. The E-50S particle has defective association properties and is incapable of peptide bond formation. It can be converted to an active 50 S ribosomal subunit when ethionine-treated bacteria are incubated under conditions which permit methylation of submethylated cellular components (presence of methionine) in the absence of de novo protein and RNA synthesis (presence of rifampicin).Total reconstitution of 50 S ribosomal subunits in vitro using normal 23 S and 5 S ribosomal RNA and proteins prepared from E-50S particles yields active subunits only if L16 is also added. The hypothesis that E-50S particles accumulate in ethionine-treated bacteria because the absence of methylation of one or more of their components blocks a late stage (L16 integration) in the normal 50 S assembly process is discussed.     

Proteins fro Escherichia coli ribosomes involved in the binding of erythromycin.

When 50 S subunits from Escherichia coli ribosomes were incubated with 1·3 m-LiC1 the resulting 1·3c core was inactive both with respect to peptidyltransferase activity and erythromycin binding (tested by equilibrium dialysis). Reconstitution experiments with purified proteins from the corresponding split fraction SP1·3 revealed that only L16 (reconstituted with the 1·3c core in a tenfold excess) could restore high activity in both systems.When 30 out of the 34 isolated ribosomal proteins were tested directly for binding or erythromycin, L15 was able to bind the drug, in contrast to all other proteins including L16. Total reconstitution experiments with the 50 S subunit demonstrated an absolute requirement for L15 and L16 with respect to both drug binding and peptidyltransferase activity.     

Molecular interactions between ribosomal proteins: a study of the S6-S18 interaction.

The proteins S6 and S18 from the 30 S ribosomal subunit of Escherichia coli were isolated to a purity of greater than 95%, characterized in solution, and investigated by sedimentation equilibrium for possible intermolecular interactions in a dilute salt reconstitution buffer. It was observed that neither protein S6 nor S18 has a tendency to self-associate in the concentration range studied. An analysis of solution mixtures containing proteins S6 and S18 revealed a species of molecular weight greater than either of the proteins. Proteins S6 and S18 were found to interact with an equilibrium constant of association of 6.6 ± 4.2 × 10⁴m^?1 at 3 °C with a Gibbs free energy of interaction, ΔG ° = ?6.1 kcal/mol. These data are part of those collected to help in building a map of the energetics in the 30 S ribosomal subunit, which provides for the stabilization of the structure.     

Interactions between viomycin resistance and streptomycin resistance on ribosomes of Mycobacterium smegmatis.

We have investigated the mechanism of the expression of resistance to high levels of viomycin and coresistance to streptomycin in a mutant strain of Mycobacterium smegmatis ATCC 14468 (AC-13) which was obtained by serial transfers of parental cells to media containing increasing concentrations of viomycin. It was shown previously that resistance to viomycin by strain AC-13 was due to an alteration in the 50 S ribosomal subunit (20). However, genetic analysis has shown that mutation in 50 S subunits alone gave only low level resistance to viomycin. When a streptomycin resistant mutation (caused by an alteration in the 30 S subunit) was introduced into the low level viomycin resistant recombinant strains, most of them were highly resistant to viomycin. Some recombinants were resistant to intermediate levels of viomycin, and the remainder were not affected by the introduction of the str^r allele. Studies with in vitro cell-free systems have shown that streptomycin resistant 30 S ribosomal subunits obtained from a high level viomycin resistant recombinant were able to modify the levels of resistance to viomycin expressed by the 50 S ribosomal subunit. These findings provide additional evidence concerning the functional relationship between 30 S and 50 S ribosomal components in ribosomes.     

Ribosomal proteins: XLIII. In vivo assembly of Escherichia coli ribosomal proteins

Isolation of ribosomal precursors from Escherichia coli K12 is described. The RNA and protein content of the precursor particles was determined.One physiologically stable precursor was found for the 30 S subunit. The assembly scheme is as follows: p16 S RNA + 9 proteins → p30 S (“21 S” precursor) p30 S + 12 proteins → 30 S subunit where p is precursor.Each of the two precursors for the 50 S subunit, P₁50 S and p₂50 S (“32 S” and “43 S” precursors, respectively), contains p5 S + p23 S RNA's in a 1:1 molar ratio. The assembly scheme is as follows: p23 S RNA + p5 S RNA + 16 or 17 proteins → p₁50 S

In contrast to the p₂50 S precursor the p₁50 S precursor is not similar to any core particles, which were obtained by treating 50 S subunits with different concentrations of LiCl or CsCl.The precursors p30 S and p₂50 S can be converted into active 30 S and 50 S sub-units, respectively, by incubation at 42 °C in the presence of ribosomal proteins and under RNA methylating conditions.     

Mitochondrial ribosome assembly in Neurospora crassa. Chloramphenicol inhibits the maturation of small ribosomal subunits.

Recent results suggest that, in Neurospora crassa, one small subunit mitochondrial ribosomal protein (S-4a, M_r 52,000) is synthesized intramitochondrially (Lambowitz et al., 1976). We now find that, when wild-type cells are treated with chloramphenicol to block mitochondrial protein synthesis, the maturation of 30 S mitochondrial ribosomal subunits is rapidly inhibited and there is an accumulation of CAP-30 S particles which sediment slightly behind mature small subunits. Electrophoretic analysis suggests that the CAP-30 S particles are deficient in several proteins including S-4a and that they are enriched in a precursor RNA species that is slightly longer than 19 S RNA. Chloramphenicol also appears to inhibit the maturation of 50 S ribosomal subunits, but this effect is much less pronounced. Continued incubation in chloramphenicol leads to a decrease in the proportion of total mitochondrial ribonucleoprotein present as monomers, possibly reflecting the depletion of competent subunits. After long-term (17 h) growth in chloramphenicol, mitochondrial ribosome profiles from wild-type cells show decreased ratios of small to large subunits, a feature which is also characteristic of the poky (mi-1) mutant. Pulse-labeling experiments combined with electrophoretic analysis show that the synthesis of mitochondrial ribosomal RNAs is relatively unaffected by chloramphenicol and that, despite the deficiency of small subunits, 19 S and 25 S RNA are present in normal ratios in whole mitochondria. By contrast, 19 S RNA in poky mitochondria is rapidly degraded leading to a decreased ratio of 19 S to 25 S RNA. The significance of these results with respect to the etiology of the poky mutation is discussed and a model of mitochondrial ribosome assembly that incorporates all available data is presented.     

A study of E. coli and T. maritima ribosomes by atomic force microscopy

Whole 70S ribosomes and 50S and 30S ribosomal subunits of E. coli and T. maritima were studied by atomic force microscopy. Adsorption of the ribosomal subunits on a substrate revealed considerable heterogeneity of their structures. Analysis of the geometric size of the particles demonstrated essential difference between the heights of E. coli and T. maritima ribosomes 9.4 ± 0.01 nm and 10.35 ± 0.02 nm, respectively. Presumably, the difference in size is determined by the difference in organization of the mobile ribosomal domain, the L7/L12 stalk.     

Letter: Conformational changes in ribosomal subunits following dissociation of the Escherichia coli 70 S ribosome

The reactivity of various Escherichia coli ribosomal proteins with N-ethylmaleimide has been used as a probe for ribosomal topography changes during the subunit-70 S transition. With the 70 S ribosome there are several proteins from both subunits which do not react with N-ethylmaleimide, but which do so after dissociation of the 70 S particle to free 30 S and 50 S subunits. The kinetics of their exposure is slow relative to that of the 70 S dissociation reaction suggesting conformational changes in both subunits subsequent to 70 S particle dissociation.     

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

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

The RimP Protein Is Important for Maturation of the 30S Ribosomal Subunit

The in vivo assembly of ribosomal subunits requires assistance by auxiliary proteins that are not part of mature ribosomes. More such assembly proteins have been identified for the assembly of the 50S than for the 30S ribosomal subunit. Here, we show that the RimP protein (formerly YhbC or P15a) is important for the maturation of the 30S subunit. A rimP deletion (ΔrimP135) mutant in Escherichia coli showed a temperature-sensitive growth phenotype as demonstrated by a 1.2-, 1.5-, and 2.5-fold lower growth rate at 30, 37, and 44 °C, respectively, compared to a wild-type strain. The mutant had a reduced amount of 70S ribosomes engaged in translation and showed a corresponding increase in the amount of free ribosomal subunits. In addition, the mutant showed a lower ratio of free 30S to 50S subunits as well as an accumulation of immature 16S rRNA compared to a wild-type strain, indicating a deficiency in the maturation of the 30S subunit. All of these effects were more pronounced at higher temperatures. RimP was found to be associated with free 30S subunits but not with free 50S subunits or with 70S ribosomes. The slow growth of the rimP deletion mutant was not suppressed by increased expression of any other known 30S maturation factor.     

Binding of [14C]tuberactinomycin O, an antibiotic closely related to viomycin, to the bacterial ribosome.

The binding of [¹⁴C]tuberactinomycin O, an antibiotic closely related to viomycin, to $\text{E. coli}$ ribosomes has been examined by equilibrium dialysis method. The antibiotic has been observed to bind to the 70S ribosome, which possesses two binding sites: one on the 30S ribosomal subunit and another on the 50S subunit. The affinity for the large subunit is greater than that for the small subunit. The binding to both ribosomal subunits is reversed by viomycin, indicating that tuberactinomycin O and viomycin have the same binding sites on the ribosome. The results seem to be in accordance with the previous finding that viomycin exhibits dual actions on ribosomal function: the inhibition of fMet-tRNA_F (initiation) and inhibition of translocation of peptidyl-tRNA.     

E. coli HflX interacts with 50S ribosomal subunits in presence of nucleotides

HflX is a GTP binding protein of unknown function. Based on the presence of the hflX gene in hflA operon, HflX was believed to be involved in the lytic-lysogenic decision during phage infection in Escherichia coli. We find that E. coli HflX binds 16S and 23S rRNA - the RNA components of 30S and 50S ribosomal subunits. Here, using purified ribosomal subunits, we show that HflX specifically interacts with the 50S. This finding is in line with the homology of HflX to GTPases involved in ribosome biogenesis. However, HflX-50S interaction is not limited to a specific nucleotide-bound state of the protein, and the presence of any of the nucleotides GTP/GDP/ATP/ADP is sufficient. In this respect, HflX is different from other GTPases. While E. coli HflX binds and hydrolyses both ATP and GTP, only the GTP hydrolysis activity is stimulated by 50S binding. This work uncovers interesting attributes of HflX in ribosome binding.     

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.

     

Genetic studies of the ribosomal proteins in Escherichia coli. 8. Mapping of ribosomal protein components by intergeneric mating experiments between Serratia marcescens and Escherichia coli

Summary Ribosomal protein compositions of Serratia marcescens and Escherichia coli K12 were analyzed by using carboxymethyl cellulose column chromatography. Nine 50S and nine 30S ribosomal proteins of E. coli K12 could be distinguished from those of S. marcescens on the chromatogram.Episomes of E. coli K12, which cover the streptomycin(str) region of the chromosome, were transferred to S. marcescens. Chromatographic analyses were made on the ribosomal proteins extracted from these hybrid strains. At least nine 30S and six 50S ribosomal proteins of E. coli-type could be detected in the ribosomes of the hybrid strains in addition to the ribosomal proteins of S. marcescens.     

Ribosomal distribution in a polyamine auxotroph of Escherichia coli.

The distribution of ribosomal particles has been studied in a polyamine-deficient mutant of Escherichia coli by sucrose gradient centrifugation analysis. Lysates from starved cells contained less 70S monomers and 30S subunits but more 50S particles than those prepared from bacteria supplemented with putrescine. The addition of the polyamine to putrescine-depleted cells induced a rapid change of the ribosomal profile. A similar effect could be obtained in vitro by equilibrium dialysis against a polyamine-containing solution. The ribosomal pattern obtained from starved bacteria was specific for polyamine deficiency. We conclude that the changes in ribosomal profiles upon restoration of putrescine levels in previously starved cells denote a shift of the equilibrium between 30S-50S couples and ribosomal subunits.     

Ribosomal assembly deficiency in an Escherichia coli thermosensitive mutant having an altered L24 ribosomal protein.

Localized P1 mutagenesis was used to screen for conditionally lethal mutations in ribosomal protein genes. One such mutation, 2859mis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and cotransduces at 98% with rpsE (S5). The 2869mis mutation leads to thermosensitivity and impaired assembly in vivo of 50 S ribosomal particles at 42 °C. The strain carrying the mutation has an altered L24 ribosomal protein which at 42 °C shows weaker affinity for 23 S RNA than the wild-type protein. The mutational alteration involves a replacement of glycine by aspartic acid in protein L24 from the mutant. We conclude therefore that the 2859mis mutation affects the structural gene for protein L24 (rplX).     

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.     

Site of synthesis of spinach chloroplast ribosomal proteins and formation of incomplete ribosomal particles in isolated chloroplasts

Chloroplast ribosomal proteins from spinach have been prepared in the presence of a protease inhibitor and some modifications have been introduced to the previous characterization of the 50S subunits (Mache et al., MGG, 177, 333, 1980): 33 ribosomal proteins are detected instead of 34. No change has been observed for the 30S subunits.Using a light-driven system of protein synthesis it is shown that up to ten ribosomal proteins of the 30S and eight proteins of the 50S subunits are made in the chloroplast.Newly synthesized ribosomal subunits have been analysed on CsCl gradients after sedimentation at equilibrium, allowing the separation of fully assembled subunits from incomplete ribosomal particles. Most of the newly made 50S subunits are fully assembled (=1.634). A small amount of incomplete 50S particles (=1.686) is detectable. Newly made 30S subunits (=1.598) and incomplete 30S particles (=1.691) are also observed. The ribosomal proteins of the incomplete 30S have been determined. They contain eight or nine of the 30S-proteins, seven of which are synthesized within the chloroplast. It is suggested that incomplete ribosomal particles resulted from a step in the assembly of ribosomal subunits.     

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.