Peptidyltransferase activity of ribosomes and a ribosome precursor from a mutant of Escherichia coli.

Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that caused the accumulation of ribonucleoprotein ('47S') particles during exponential growth. These particles are precursors to 50S ribosomes that lack three ribosomal proteins. Peptidyltransferase activity and binding at the peptidyl site of the peptidyltransferase centre are greatly decreased in 47S particles. Both these activities are lower in the 50S and 70S ribosomes of strain 15-28 than in its parent. Unusual assembly of the larger ribosomal subunit in strain 15-28 may produce completed ribosomes with diminished biological activity.     

An unusual precursor of 50S ribosomes in a mutant of Escherichia coli.

Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that leads to the accumulation of large amounts of ribonucleoprotein ("47S") particles during exponential growth. These particles are precursors to 50S ribosomes, but are distinct from precursors detected by pulse-labelling of the parent strain and also from ribosome precursors that accumulate during inhibition of growth by CoC12. Either ribosome assembly in the mutant differs from that in the wild-type strain, or 47S particles represent a hitherto unstudied stage in the synthesis of 50S ribosomes.     

The composition of an unusual precursor of 50 S ribosomes in a mutant of Escherichia coli.

Escherichia coli strain 15--28 is a mutant which during exponential growth contains large amounts of a '47S' ribonucleoprotein precursor to 50S ribosomes. The '47S particles' are more sensitive to ribonuclease than are 50S ribosomes. The 23 S RNA of 47S particles may be slightly undermethylated, but cannot be distinguished from the 23S RNA of 50S ribosomes by sedimentation or electrophoresis. Isolated particles have 10--15% less protein than do 50S ribosomes; proteins L16, L28 and L33 are absent. Comparison with precursor particles studied by other workers in wild-type strains of E. coli suggests that the assembly of 50S ribosomes in strain 15--28 is atypical.     

Intermediates in the assembly of ribosomes by a mutant of Escherichia coli.

Escherichia coli strain 15--28 is a mutant that accumulates ribonucleoprotein ('47 S') particles during exponential growth. These particles contain mature 23 S rRNA, but lack three of the proteins of the larger ribosomal subunit, to which they are a precursor. In organisms growing at 20 degrees C, assembly of 47 S particles involves three intermediates that contain precursor 23 S rRNA, one of which has the same sedimentation properties as 47 S particles. Assembly of 50 S ribosomal subunits in the parent strain is 'normal'. There are three intermediates; each contains precursor 23 S rRNA, and one cannot be distinguished from completed subunits by sedimentation. Synthesis of 30 S ribosomal subunits in parent and mutant strains is qualitatively similar, but quantitatively different. When growth is at 37 degrees C, assembly in the mutant alters. There are now two sequential precursors to 47 S particles. Both contain precursor 23 S rRNA; one has the same sedimentation coefficient as 47 S particles. In some respects, synthesis in the mutant proceeds as though 47 S particles, rather than 50 S ribosomal subunits, are the end-product of assembly.     

Stoichiometry and structure of a complex of acidic ribosomal proteins

The acidic proteins B-L13 (homologous to Escherichia coli protein L7/L12) and B-L8, from the 50 S subunit of Bacillus stearothermophilus ribosomes, form a stable complex. Trypsin digestion of ribosomes generates an N-terminal fragment of B-L13 (approximately residues 1 to 47) which can associate with B-L8, displacing intact B-L13, and bind to B-L13-deficient ribosomes. Displacement of B-L13 from the B-L8 · B-L13 complex by the B-L13 N-terminal fragment causes a change in gel electrophoretic mobility of the complex, and titration of the complex with fragment indicates unambiguously that it contains four molecules of B-L13. Evidence is presented that B-L13 forms a dimer in solution, and that the dimer associates intact with B-L8. Reconstituted 50 S subunits in which B-L13 is replaced by its N-terminal fragment have the same functional properties as 50 S subunits missing B-L13 altogether: polypeptide synthesis is reduced but not abolished; ability to bind elongation factor EF-G and GTP is severely reduced; and peptidyl transferase activity and ability to associate with a 30 S subunit · Phe-tRNA · poly(U) complex are unaffected (relative to intact 50 S subunits).     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

Ribosomal protein synthesis by a mutant of Escherichia coli

The mutant strain of Escherichia coli, TP28, synthesises ribosomes by an abnormal pathway and accumulates large quantities of 47S ribonucleoprotein particles. The protein complement of mutant 70S ribosomes is normal but 47S particles contain only traces of proteins L28 and L33 and have a significantly reduced content of four other proteins. The mutation reduces the rates of synthesis of L28 and L33 by about half but other widespread alterations ensue. In particular, ribosomal protein synthesis in the mutant strain becomes less well balanced than in its parent: some proteins, particularly those from promoter-proximal genes, are oversynthesized and their excess then degraded.     

Interaction between the erythromycin and chloramphenicol binding sites on the Escherichica coli ribosome.

The effects of chloramphenical on the binding kinetics of a fluorescein isothiocyanate derivative of 9(S)-erythromycylamine with 70S and 50S ribosomes have been studied by direct fluorimetric measurements. While chloramphenicol had little effect on the second-order 70S binding rate of the erythromycin analogue, it substantially reduced the dissociation rate of the fluorescent antibiotic-70S ribosome complex. This could be explained by simultaneous binding of both antibiotics to the 70S ribosome. The kinetic results suggest that chloramphenicol-saturated 70S particles bind the erythromycin analogue four times stronger and this was confirmed by direct binding studies. In additon, chloramphenicol causes a twofold increase in the intrinsic fluorescence of the 70S-bound analogue. This increase in fluorescence was used to study the kinetics of chloramphenicol binding to 70S ribosomes containing the fluorescent derivative. The fluorescence change followed first-order kinetics, suggesting that chloramphenicol induces a conformational change in the 70S particle. This could explain both its effect on erythromycin binding and on the fluorescence of bound analogue. Less detailed results with the 50S particle indicate a qualitively similar picture of erythromycin-chloramphenicol interactions.     

Two monoclonal antibodies against Escherichia coli ribosomal protein L2 distinguish different locations for their respective epitopes in intact ribosomes

Two monoclonal antibodies raised against intact Escherichia coli ribosomal protein L2 were isolated, affinity-purified, and characterized. One of the antibodies (Ab 5-186) recognizes an epitope within residues 5-186, and the other (Ab 187-272) recognizes an epitope within residues 182-272. Both antibodies strongly inhibit in vitro polyphenylalanine synthesis when they are first allowed to bind to 50 S subunits prior addition of 30 S subunits. However, only Ab 187-272 is inhibitory when added to preformed 70 S ribosomes. Ab 5-186 binds to 50 S subunits but not to 70 S ribosomes. Ab 187-272 does not cause dissociation of 70 S ribosomes under the ionic conditions of the assay for polyphenylalanine synthesis (15 mM magnesium), although at 10 mM magnesium it does cause dissociation. Both antibodies inhibit the reassociation of 50 S with 30 S subunits. Both antibodies strongly inhibit peptidyltransferase activity. The two antibodies differ in their effects on interactions with elongation factors Tu (EF-Tu) and G (EF-G). Neither antibody significantly inhibits EF-G-dependent GTPase activity, nor the binding of EF-G when the antibodies are incubated with 50 S subunits; however, Ab 187-272 causes a decrease in the binding of EF-Tu X aminoacyl-tRNA X GTP ternary complex and of EF-Tu-dependent GTPase when it is incubated with 70 S ribosomes. The Fab fragments of both antibodies had effects similar to the intact antibodies. The results show that monoclonal antibodies can be used to discriminate different regions of L2 and that EF-Tu and EF-G do not have identical ribosomal binding sites.     

Evidence for a tRNA/rRNA interaction site within the peptidyltransferase center of the Escherichia coli ribosome

A nine-base oligodeoxyribonucleotide complementary to bases 2497-2505 of 23S rRNA was hybridized to both 50S subunits and 70S ribosomes. The binding of the probe to the ribosome or ribosomal subunits was assayed by nitrocellulose filtration and by sucrose gradient centrifugation techniques. The location of the hybridization site was determined by digestion of the rRNA/cDNA heteroduplex with ribonuclease H and gel electrophoresis of the digestion products, followed by the isolation and sequencing of the smaller digestion fragment. The cDNA probe was found to interact specifically with its rRNA target site. The effects on probe hybridization to both 50S and 70S ribosomes as a result of binding deacylated tRNA(Phe) were investigated. The binding of deacylated tRNA(Phe), either with or without the addition of poly(uridylic acid), caused attenuation of probe binding to both 50S and 70S ribosomes. Probe hybridization to 23S rRNA was decreased by about 75% in both 50S subunits and 70S ribosomes. These results suggest that bases within the 2497-2505 site may participate in a deacylated tRNA/rRNA interaction.     

Abnormal ribosome assembly in a mutant of Escherichia coli.

The mutant strain, 15--28, of Escherichia coli accumulates ribonucleoprotein ('47S') particles that were previously shown [Markey, Sims & Wild (1976) Biochem. J. 158, 451--456] to be an unusual intermediate in the assembly of 50S ribosomal subunits...     

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.     

Studies on native ribosomal subunits from rat liver. Evidence for activities associated with native 40S subunits that affect the interaction with acetylphenylalanyl-tRNA, methionyl-tRNAf, and 60S subunits.

The binding of the initiator tRNA Met-tRNAf, and of acetylphenylalanyl-tRNA, has been examined with rat liver 40S subunits derived from 80S ribosomes by dissociation with native 40S subunits sedimented from the postmicrosomal fraction and with native 40S subunits extracted with high salt-containing solutions. Binding of Met-tRNAf and acetylphenylalanyl-tRNA to derived and to salt-extracted native 40S subunits is observed in the presence of the appropriate polynucleotide template and a highly purified binding factor obtain from the soluble fraction of rat liver homogenates (R.L. IF-1). Native 40S subunits bind acetylphenylalanyl-tRNA in a reaction that requires poly(U) but not exogenous binding factor; however, Met-tRNAf is not bound to native subunits, even when supplemented with the soluble binding factor, or under conditions where factor-independent, high Mg2+-stimulated binding is observed with the derived and the salt-washed native 40S subunits. The extract obtained from native 40S subunits promotes the binding of acetylphenylalanyl-tRNA but not Met-tRNAf to derived and to salt-extracted native subunits. The addition of native 40S extract to incubations containing R.L. IF-1, Met-tRNAf, and derived 40S subunits, inhibits the formation of 40S-Met-tRNAf complex. These data suggest that the binding activity that is specific for 40S subunits and initiator tRNA, and an activity that inhibits the interaction with Met-tRNAf specifically, are both associated with native 40S subunits, and can be extracted from them by treatment with high salt-containing solutions. Derived 40S subunits react quantitatively with 60S particles to form 80S ribosomes which do not bind acetylphenylalanyl-tRNA with binding factor R.L. IF-1. Native 40S subunits react only partly with 60S subunits; about half of the native 40S subunit population forms 80S ribosomes which do not subsequently bind acetylphenylalanyl-tRNA; the remaining native 40S subunits which do not react with 60S particles bind acetylphenylalanyl-tRNA but to a lesser extent. When preformed native 40S-acetylphenylalanyl-tRNA complex is incubated with 60S subunits, about half of the subunits form an 80S-acetylphenylalanyl-tRNA complex, while the rest remains as 40S-acetylphenylalanyl-tRNA. The addition of native 40S subunit salt extract to incubations containing preformed 80S ribosomes dissociates the particles to subunits. These data suggest that in addition to the initiator tRNA binding activity and the activity that inhibits Met-tRNAf interaction, part of the native 40S subunit population also contains an activity that dissociates 80S ribosomes.     

Identification of a rRNA/chloramphenicol interaction site within the peptidyltransferase center of the 50 S subunit of the Escherichia coli ribosome.

We have used oligodeoxyribonucleotide probes to investigate possible interactions between chloramphenicol and portions of the rRNA contained within the peptidyltransferase center of the Escherichia coli ribosome. Oligodeoxyribonucleotide probes complementary to bases 2448-2454, 2468-2482, and 2497-2505 of 23 S rRNA were hybridized to 50 S subunits in situ. Probe binding was qualitatively assessed by sucrose gradient centrifugation. Each probe was shown to bind specifically with its intended binding site through digestion of the rRNA within the RNA/DNA hetero-duplexes with RNase H and analysis of the digestion fragments using gel electrophoresis. Competitive binding experiments were conducted between each probe and the antibiotics chloramphenicol and erythromycin. The binding of a probe complementary to bases 2497-2505 was attenuated by 70% upon the binding of chloramphenicol. A probe complementary to bases 2468-2482 showed an increase in binding of 14% while binding of a probe complementary to bases 2448-2454 was not affected by chloramphenicol binding. Erythromycin did not affect the binding of any of these probes to 50 S subunits. These results suggest that bases within the 2497-2505 region of 23 S rRNA in E. coli may be involved in a chloramphenicol/rRNA interaction.     

The in vitro binding of virginiamycin M to bacterial ribosomes and ribosomal subunits

Summary Virginiamycin M (VM), an antibiotic of type A synergimycin group of antibiotics, binds to bacterial ribosomes and subunits in vitro: the amount of linked drug is linearly dependent on ribosome and VM concentrations. The technique used to measure the association reaction is based on the finding that the unbound drug is adsorbed by norite A: this procedure is twice as sensitive as the sedimentation and filtration methods (for technical reasons, column chromatography and equilibrium dialysis are unsuitable for this study). Saturation curves with 70S and 50S particles overlap, thus indicating a comparable affinity of the inhibitor for ribosomes and large subunits; instead, very small amount of VM, if any, attaches to 30S particles. Kinetics of binding is influenced by the temperature; the 4° C and 25° C saturation curves overlap, however, upon pre-incubation of ribosomes in 10 mM Mg buffer at 37° C (reactivation). This suggests that binding of VM depends on the configura tion of the 50S particles, which is altered at low temperature. Differences in Mg⁺⁺ concentration in the range 1 to 20 mM do not modify the binding curve, nor does the replacement of K⁺ by either NH ₄ ⁺ or Na⁺. Previously bound labelled VM is slowly displaced by an excess of unlabeled VM, and the associa tion curve remains unchanged in the presence of VS. Binding of VM is inhibited (10 to 60%) in the presence of an excess (tenfold to hundredfold) of one of the 50S inhibitors: chloramphenicol, oleandomycin and erythromycin. From the Scatchard plot, an as sociation constant of 3.2 × 10⁵M^–1 has been calculated: this value is about 1/8 of that reported for VS, a component of type B synergimycin group of antibiotics. The v value is 0.85 for both ribosomes and large subunits, indicating a monomolecular association of VM with ribonucleoprotein particles.     

Conformational studies of Escherichia coli ribosomes with the use of acridine orange as a probe

The interaction of E. coli vacant ribosomes with acridine orange (AO) was studied, to obtain conformational information about rRNAs in ribosomes. Acridine orange binds to an RNA in two different modes: cooperative outside binding with stacking of bound AO's and intercalation between nucleotide bases. Free 16S and 23S rRNAs have almost identical affinities to AO. At 1 mM Mg2+, AO can achieve stacking binding on about 40% of rRNA phosphate groups. The number of stacking binding sites falls to about 1/3 in the 30S subunit in comparison with free 16S rRNA. In the 50S subunit, the number of stacking binding sites is only 1/5 in comparison with free 23S rRNA. Mg2+ ions are more inhibitory for the binding of AO to ribosomes than to free rRNAs. The strength of stacking binding appears to be more markedly reduced by Mg2+ in active ribosomes than in rRNAs. "Tight couple" 70S particles are less accessible for stacking binding than free subunits. The 30S subunits that have irreversibly lost the capability for 70S formation under low Mg2+ conditions have an affinity to AO that is very different from that of active 30S but similar to that of free rRNA, though the number of stacking binding sites is little changed by the inactivation. 70S and 30S ribosomes with stacking bound AO's have normal sedimentation constants, but the 50S subunits reversibly form aggregates.     

Construction and functional analysis of ribosomal 5S RNA from Escherichia coli with single base changes in the ribosomal protein binding sites

The ribosomal 5S RNA gene from E. coli was altered by oligonucleotide-directed mutagenesis at positions A66 and U103. The mutant genes were cloned into an expression vector and selectively transcribed in an UV-sensitive E. coli strain using a modified maxicell system. The mutant 5S RNA genes were found to be transcribed and processed normally. The 5S RNA molecules were assembled into 50S ribosomal subunits. Under in vitro conditions the stability of the mutant 70S ribosomes seemed, however, to be reduced, since they dissociated into their subunits more easily than those of the wild type. The isolated mutated 5S RNAs with base changes in the ribosomal protein binding sites for L18 and L25, together with a point mutant at G41 (G to C), constructed earlier, were tested for their capacity to bind the 5S RNA binding proteins L5, L18 and L25. The following effects were observed: The base change A66 to C within the L18 binding site did not affect the binding of the ribosomal protein L18 but enhanced the stability of the L25-5S RNA complex considerably. The base changes U103 to G and G41 to C slightly reduced the binding of L5 and L25 whereas the binding of L18 to the mutant 5S RNAs was not altered. In addition 70S ribosomes with the single point mutations in their 5S RNAs were tested in their tRNA binding capacity. Mutants containing a C41 in their 5S RNA showed a reduction in the poly(U)-dependent Phe-tRNA binding, whereas the mutations to C66 and G 103 lead to completely inactive ribosomes in the same assay. Based on previous results a spatial model of the 5S RNA molecule is presented which is consistent with the findings reported in this paper.     

A Comparison of the Native and Derived 30S and 50S Ribosomes of Escherichia coli

Four types of ribosomes occurring in E. coli have been separated by sucrose gradient centrifugation. These are the 30S and 50S particles occurring in E. coli extracts (native particles), and the 30S and 50S particles which are the subunits of 70S ribosomes (derived particles). Two criteria were used in comparing these particles: (1) The type of RNA contained in each, as determined by sedimentation velocity in the analytical ultracentrifuge. (2) The ability of mixtures of 30S and 50S ribosomes (derived 30S + derived 50S, native 30S + native 50S) to undergo the reaction: [Formula: see text] Native and derived 30S particles were found to contain 16S RNA. Derived 50S particles contained 23S RNA and a small amount of 15 to 20S RNA, whereas native 50S ribosomes contained only 16S RNA. Derived 30S and 50S particles combined to form 70S particles. However, under identical conditions, native 30S and 50S particles did not form 70S ribosomes.     

Probing ribosome function and the location of Escherichia coli ribosomal protein L5 with a monoclonal antibody

A monoclonal antibody specific for Escherichia coli ribosomal protein L5 was isolated from a cell line obtained from Dr. David Schlessinger. Its unique specificity for L5 was confirmed by one- and two-dimensional electrophoresis and immunoblotting. The antibody recognized L5 both in 50 S subunits and 70 S ribosomes. Both antibody and Fab fragments had similar effects on the ribosome functions tested. Antibody bound to 50 S subunits inhibited their reassociation with 30 S subunits at 10 mM Mg2+ but not 15 mM, the concentration present for in vitro protein synthesis. The 70 S couples were not dissociated by the antibody. The antibody caused inhibition of polyphenylalanine synthesis at molar ratios to 50 S or 70 S particles of 4:1. The major inhibitory effect was on the peptidyltransferase reaction. There was no effect on either elongation factor binding or the associated GTPase activities. The site of antibody binding to 50 S was determined by electron microscopy. Antibody was seen to bind beside the central protuberance or head of the particle, on the side away from the L7/L12 stalk, and on or near the region at which the 50 S subunit interacts with the 30 S subunit. This site of antibody binding is fully consistent with its biochemical effects.     

30S ribosomal subunits can be assembled in vivo without primary binding ribosomal protein S15

Assembly of 30S ribosomal subunits from Escherichia coli has been dissected in detail using an in vitro system. Such studies have allowed characterization of the role for ribosomal protein S15 in the hierarchical assembly of 30S subunits; S15 is a primary binding protein that orchestrates the assembly of ribosomal proteins S6, S11, S18, and S21 with the central domain of 16S ribosomal RNA to form the platform of the 30S subunit. In vitro S15 is the sole primary binding protein in this cascade, performing a critical role during assembly of these four proteins. To investigate the role of S15 in vivo, the essential nature of rpsO, the gene encoding S15, was examined. Surprisingly, E. coli with an in-frame deletion of rpsO are viable, although at 37 degrees C this DeltarpsO strain has an exaggerated doubling time compared to its parental strain. In the absence of S15, the remaining four platform proteins are assembled into ribosomes in vivo, and the overall architecture of the 30S subunits formed in the DeltarpsO strain at 37 degrees C is not altered. Nonetheless, 30S subunits lacking S15 appear to be somewhat defective in subunit association in vivo and in vitro. In addition, this strain is cold sensitive, displaying a marked ribosome biogenesis defect at low temperature, suggesting that under nonideal conditions S15 is critical for assembly. The viability of this strain indicates that in vivo functional populations of 70S ribosomes must form in the absence of S15 and that 30S subunit assembly has a plasicity that has not previously been revealed or characterized.