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.     

The pseudouridine synthase RluD is required for normal ribosome assembly and function in Escherichia coli

RluD is the pseudouridine synthase responsible for the formation of Psi1911, Psi1915, and Psi1917 in Escherichia coli 23S rRNA. Previous work from our laboratory demonstrated that disruption of the rluD gene and/or loss of the pseudouridine residues for which it is responsible resulted in a severe growth phenotype. In the current work we have examined further the effect of the loss of the RluD protein and its product pseudouridine residues in a deletion strain lacking the rluD gene. This strain exhibits defects in ribosome assembly, biogenesis, and function. Specifically, there is a deficit of 70S ribosomes, an increase in 50S and 30S subunits, and the appearance of new 62S and 39S particles. Analysis of the 39S particles indicates that they are immature precursors of the 50S subunits, whereas the 62S particles are derived from the breakdown of unstable 70S ribosomes. In addition, purified mutant 70S ribosomes were found to be somewhat less efficient than wild type in protein synthesis. The defect in ribosome assembly and resulting growth phenotype of the mutant could be restored by expression of wild-type RluD and synthesis of Psi1911, Psi1915, and Psi1917 residues, but not by catalytically inactive mutant RluD proteins, incapable of pseudouridine formation. The data suggest that the loss of the pseudouridine residues can account for all aspects of the mutant phenotype; however, a possible second function of the RluD synthase is also discussed.     

Binding of chloramphenicol and a fragment of aminoacyl-transfer ribonucleic acid to ribosomes and a ribosome precursor from a mutant of Escherichia coli.

During exponential growth, the mutatn strain Escherichia coli 15-28 accumulates 47S particles, which are unusual precursors to 50S ribosomal subunits. The 47S particles have little ability to bind chloramphenicol, but binding of a fragment of aminoacyl-tRNA is about half that by completed subunits. The 70S (and 50S) ribosomes of strain 15-28 and its parent (strain 15TP) do not differ in chloramphenicol binding. Although ribosomes from the mutant are less able than those from the parent to bind the fragment, this difference is not as marked as was found previously [Sims & Wild (1976) Biochem. J. 160, 721-726] for the binding of an analogue of peptidyl-tRNA and for peptidyltransferase activity. The altered activities may arise because strain 15-28 misassembles 50S subunits of altered conformation and because the few proteins that 47S patricles lack have vital functions in some of the partial reactions of protein synthesis.     

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.     

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.     

Authentic precursors to ribosomal subunits accumulate in Escherichia coli in the absence of functional DnaK chaperone

Escherichia coli dnaK-ts mutants are defective in the late stages of ribosome biogenesis at high temperature. Here, we show that the 21S, 32S and 45S ribosomal particles that accumulate in the dnaK756-ts mutant at 44 degrees C contain unprocessed forms of their 16S and 23S rRNAs (partially processed in the case of 45S particles). Their 5S rRNA stoichiometry and ribosomal protein composition are typical of the genuine ribosomal precursors found in a wild-type (dnaK+) strain. Despite the lack of a functional DnaK, a very slow maturation of these 21S, 32S and 45S particles to structurally and functionally normal 30S and 50S ribosomal subunits still occurs at high temperature. This conversion is accompanied by the processing of p16S and p23S rRNAs to their mature forms. We conclude that: (i) 21S, 32S and 45S particles are not dead-end particles, but true precursors to active ribosomes (21S particles are converted to 30S subunits, and 32S and 45S to 50S subunits); (ii) DnaK is not absolutely necessary for ribosome biogenesis, but accelerates the late steps of this process considerably at high temperature; and (iii) 23S rRNA processing depends on the stage reached in the stepwise assembly of the 50S subunit, not directly on DnaK.     

Ribosome modulation factor protects <Emphasis Type="Italic">Escherichia coli</Emphasis> during heat stress,but this may not be dependent on ribosome dimerisation

The role of ribosome modulation factor (RMF) in protecting heat-stressed Escherichia coli cells was identified by the observation that cultures of a mutant strain lacking functional RMF (HMY15) were highly heat sensitive in stationary phase compared to those of the parent strain (W3110). No difference in heat sensitivity was observed between these strains in exponential phase, during which RMF is not synthesised. Studies by differential scanning calorimetry demonstrated that the ribosomes of stationary-phase cultures of the mutant strain had lower thermal stability than those of the parent strain in stationary phase, or exponential-phase ribosomes. More rapid breakdown of ribosomes in the mutant strain during heating was confirmed by rRNA analysis and sucrose density gradient centrifugation. Analyses of ribosome composition showed that the 100S dimers dissociated more rapidly during heating than 70S particles. While ribosome dimerisation is a consequence of the conformational changes caused by RMF binding, it may not therefore be essential for RMF-mediated ribosome stabilisation.Abbreviations DSC Differential scanning calorimetry - MRD Maximum recovery diluent - RMF Ribosome modulation factor     

Reversion from erythromycin dependence in Escherichia coli: strains altered in ribosomal sub-unit association and ribosome assembly

A mutant of Escherichia coli dependent on erythromycin for growth spontaneously gives erythromycin-independent strains with altered or missing ribosomal proteins. strains with defects in ribosome assembly were sought and obtained from among these revertants. Two organisms in which ribosomal protein L19 is altered and absent respectively have 70S ribosomes whose dissociation into sub-units is particularly sensitive to pressures generated during centrifuging. The mutant that lacks protein L19 also accumulates ribosome precursor particles during exponential growth as do others including mutants that lack proteins S20 or L1. These strains also show unbalanced synthesis of RNA and so will be useful in investigating both the pathways and the regulation of ribosome assembly.     

Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotropic, streptomycin-resistant mutants of Escherichia coli.

Some of the spontaneous streptomycin-resistant mutants of Escherichia coli strain C600 exhibit pleiotropic effects in addition to the antibiotic resistance. These effects include decreased growth rates, reduced levels of certain enzymes, and poor support of bacteriophage growth. One of these mutants, strain SM3, was studied further. We have examined the question of whether the reduced growth rate of the mutant SM3 is related to the reduction in relative amounts of ribosomes or to the reduction in the efficiency of ribosomes in protein synthesis. Measurements of alpha, the differential synthesis rate of ribosomal protein, revealed that the protein synthesis effeciency of ribosomes from the mutant strain SM3 was reduced about twofold relative to that of the parent strain C600. Measurements of the induction lag for beta-galactosidase and of the synthesis time of several different molecular-weight classes of proteins indicated that the mutation resulted in a marked reduction in the peptide chain growth rate. This reduction in the chain growth rate probably accounted for most of the observed reduction in the growth rate of the mutant strain. These experimental results show that the strA gene product, the S12 protein of the 30S subunit, is involved in some aspect of protein chain elongation. Presumably this involvement occurs during the messenger ribonucleic acid-directed binding of transfer ribonucleic acid to the ribosome.     

23S rRNA甲基转移酶RrmJ促进细菌50S亚基后期组装

核糖体是所有细胞中负责蛋白质合成的分子机器。它自身在细胞内的组装成熟过程受到严密调控,需要诸多组装因子的参与。RrmJ是原核生物中一类保守的甲基转移酶,能够甲基化修饰核糖体上肽基转移酶中心（peptidyl transferase center, PTC）内A环的U2552位点。敲除rrmJ基因的大肠杆菌表现出显著的生长缺陷及50S亚基组装前体的累积,因而RrmJ在50S亚基组装中具有重要作用。本研究对细菌生长实验与核糖体图谱分析表明,回补表达RrmJ的质粒对于ΔrrmJ菌株生长缺陷有显著改善,50S前体累积现象也得到有效缓解。通过共沉淀实验证明,RrmJ与ΔrrmJ菌株中提取的50S前体结合能力显著强于缺失型或野生型菌株中纯化的成熟50S;当加入S-腺苷甲硫氨酸时,该酶与50S前体结合能力显著下降。冷冻电镜三维重构数据进一步阐明,缺失型菌株50S前体主要停滞在组装晚期两个PTC区域成熟程度不同的特定时段。综合上述结果表明,U2552位点的修饰发生在50S亚基组装晚期特定阶段,这一事件不仅会加速A环的RNA螺旋折叠,另有可能促进附近PTC区域结构成熟。     

The roles of proteins L28 and L33 in the assembly and function of Escherichia coli ribosomes in vivo

Strain BM108 of Escherichia coli has a chromosomal mutation in the rpmB , G operon that prevents synthesis of ribosomal proteins L28 and L33. The mutation was lethal unless synthesis of protein L28 was induced from a plasmid. Without protein L28, RNA and protein synthesis were linear rather than exponential. No 70S ribosomes were made. Instead, RNA accumulated in '30S material' and '47S particles'; the latter were distinct from 50S ribosomal subunits, lacked proteins L28 and L33 and had substoicheometric amounts of three other proteins. When L28 synthesis was induced (but protein L33 was still absent), the strain grew as well as, and assembled 70S ribosomes with similar kinetics to, a wild-type control. Thus, protein L28 is required for ribosome assembly in strain BM108 while protein L33 has no significant effect on ribosome synthesis or function.     

Biogenesis of mitochondria. 53

Summary As shown by gel electrophoresis analysis, E. coli mutant 219 is mutated on the gene coding for S4. This mutant and the parental strain have been studied at the permissive (30°) and the non-permissive temperature (42°) for ribosome assembly and r-protein biosynthesis.The extracts of cells grown at the non-permissive temperature were analyzed by sucrose gradients: Particles sedimenting more slowly (28S) than normal 30S accumulate while 50S precursors undergo maturation and attach to the preformed 30S subunits yielding 70S ribosomes. In addition a small but detectable amount of 30S is also synthesized at 42°. The 28S particles contain all 30S r-proteins except S1, S2 and S12; S5, S7 and S21 are present in reduced amount.The relative rate of biosynthesis of individual r-proteins was determined by pulse-labelling the cells with radioactive leucine. Individual r-proteins were purified from cell extract by the three-dimensional gel electrophoresis technique. The relative rate of biosynthesis of 50S proteins is unchanged in mutant cells grown at 42°. Only the rate of synthesis of five 30S proteins is modified by the temperature shift: S10, S13, S20 and S21 have an increased rate, while S18 is synthesized at a reduced rate. Thus in cells deficient in the assembly of 30S subunits, although the biosynthesis of a few 30S r-proteins is specifically altered, the synthesis of most r-proteins appears to be controlled in the same way as are total cell proteins.     

Identification of a Precursor Pool of Ribosome Protein in Escherichia coli

Antibodies prepared against proteins from 50S ribosomes of Escherichia coli also reacted with the supernatant proteins of a cell-free extract of E. coli which was ribosome-free. A reaction of immunological identity (Ouchterlony tests) was demonstrated for one of these supernatant proteins and one protein found in 50S ribosomes. Isotope experiments involving a shift from (14)C-leucine medium to (12)C-leucine medium showed that these proteins are not formed by breakdown of ribosomes during the preparation of cell-free extracts, but instead represent a pool of ribosome protein which is utilized during growth. In shift experiments from (14)C-leucine to (12)C-leucine medium, the kinetics of disappearance of labeled supernatant ribosome proteins (as measured by reaction with antibody) indicated that half the pool is depleted in 0.1 generation time at 37 C in glucose-salts medium. The pool was also depleted under conditions of amino acid starvation of a "relaxed" strain which accumulated "relaxed" particles. Most, if not all, of the protein present in "relaxed" particles was derived from the pool. The pool represented about 3 to 4% of the total soluble proteins in the ribosome-free supernatant fluid of an E. coli extract.     

Ribonucleic Acid and Protein Synthesis in a Mutant of Bacillus subtilis Defective in Potassium Retention

A mutant of Bacillus subtilis 168 (strain 168 KL), which had lost its normal capacity to accumulate K(+), was used to explore the interrelationship between protein and ribonucleic acid (RNA) synthesis. In contrast to the wild type, the growth rate of strain 168 KL was markedly dependent on the K(+) concentration in the medium. K(+) uptake in the mutant strain was identical to that in the parent, but the mutant was unable to retain and accumulate K(+). Protein synthesis was markedly dependent on the K(+) concentration in the medium, whereas RNA synthesis was relatively unaffected by changes in the level of K(+). Most of the RNA synthesized during K(+) depletion was ribosomal RNA; it appeared in crude extracts in the form of ribonucleoproteins particles with sedimentation values between 4S and 30S. These particles were converted into mature ribosomes when growth was allowed to resume by the addition of K(+). Simultaneous synthesis of RNA and protein was necessary for the quantitative conversion of the ribonucleoprotein particles into ribosomes. During recovery from K(+) depletion, ribosomal protein was synthesized in preference to the other proteins of the cell.     

Tests of the ribosome editor hypothesis

Summary Peptidyl-tRNA dissociates from the ribosomes of Escherichia coli during protein biosynthesis. The ribosome editor hypothesis states that incorrect peptidyl-tRNAs dissociate preferentially. Editing would therefore prevent the completion of proteins containing misincorporated amino acids. We have isolated a mutant strain of E. coli that dissociates some peptidyl-tRNAs at a fivefold lower rate than its parent strain, and that synthesizes significantly more erroneous complete proteins. This strain is also partially resistant to the antibiotic erythromycin, which in wildtype E. coli stimulates the dissociation of peptidyl-tRNA from ribosomes. The data suggest that in this mutant all peptidyl-tRNAs are bound to the ribosome more tightly than normally during protein synthesis. Because of the inverse correlation between the accuracy of synthesis of complete proteins and the rate of dissociation of peptidyl-tRNA from the ribosome, we propose that the mutant contains a defective ribosomal editor.     

The activity of ribosome modulation factor during growth of <Emphasis Type="Italic">Escherichia coli</Emphasis> under acidic conditions

Expression of the gene encoding ribosome modulation factor (RMF), as measured using an rmf-lacZ gene fusion, increased with decreasing pH in exponential phase cultures of Escherichia coli. Expression was inversely proportional to the growth rate and independent of the acidifying agent used and it was concluded that expression of rmf was growth rate controlled in exponential phase under acid conditions. Increased rmf expression during exponential phase was not accompanied by the formation of ribosome dimers as occurs during stationary phase. Nor did it appear to have a significant effect on cell survival under acid stress since the vulnerability of an RMF-deficient mutant strain was similar to that of the parent strain. Ribosome degradation was increased in the mutant strain compared to the parent strain at pH 3.75. Also, the peptide elongation rate was reduced in the mutant strain but not the parent during growth under acid conditions. It is speculated that the function of RMF during stress-induced reduction in growth rate is two-fold: firstly to prevent reduced elongation efficiency by inactivating surplus ribosomes and thus limiting competition for available protein synthesis factors, and secondly to protect inactivated ribosomes from degradation.     

Isolation of stable ribosomal subunits from spores of Bacillus cereus.

Analyses of ribosomes extracted from spores of Bacillus cereus T by a dryspore disruption technique indicated that previously reported defects in ribosomes from spores may arise during the ribosome extraction process. The population of ribosomes from spores is shown to cotain a variable quantity of free 50S subunits which are unstable, giving rise to slowly sedimenting particles in low-Mg2+ sucrose gradients and showing extremely low activity in in vitro protein synthesis. The majority of the ribosomal subunits in spores, obtained by dissociation of 70S ribosomes and polysomes, are shown to be as stable as subunits from vegetative cells, though the activity of spore polysomes was lower than that of vegetative ribosomes. In spite of the instability and inactivity of a fraction of the spore's ribosomal subunits, the activity of the total population obtained from spores by the dry disruption technique was 32% of vegetative ribosome activity, fivefold higher than previously obtained with this species. The improvement in activity and the observed variability of subunit destabilization are taken as evidence for partial degradation of spore ribosomes during extraction.     

The Synthesis of Ribosomes in E. coli: IV. The Synthesis of Ribosomal Protein and the Assembly of Ribosomes

The incorporation of C¹⁴ leucine into the protein moiety of ribosomes has been studied as a sequel to the studies of ribosomal RNA synthesis. In contrast to the latter studies, labeled leucine is incorporated directly into 50S and 30S ribosomes without measurable delay by precursor stages. There is, however, evidence of some transfer of radioactivity from the 43S group of particles to the 50S. The inhibition of protein synthesis by chloramphenicol results in the accumulation of material similar to the eosome—the primary precursor in ribosome synthesis. There is also evidence for the synthesis of some neosome. The results of the studies of ribosomal RNA and protein synthesis are combined into a model of ribosome synthesis. Finally, consideration is made of the significance of these studies of ribosome synthesis for general problems of protein synthesis and information transfer.     

Bevorzugter Einbau markierten Uridins in die Vorläufer von Chloroplasten-Ribosomen in Algenzellen

Summary After short time pulses with 5-[3H]uridine have been given to Chlorella cells, most of the radioactivity of the ribosome fractions is neither in the polysomes nor in the cytoplasmic ribosomes. Peaks with sedimentation of about 50 S and 30 S are found which are comparable in sedimentation to ribosomal subunits of Escherichia coli. During chase treatment with the one-hundred-fold amount of unlabelled uridine, the radioactivity shifts into the 70 S region. The RNA of the rapidly labelled 50 S and 30 S particles is shown to have 23 S, 14 S and 5 S, respectively.In contrast to this, radioactive inorganic phosphate and amino acids are mainly incorporated into the cytoplasmic ribosomes with 80 S and into, their polysomes.The chloroplast-damaged mutant of Chlorella, Nr.125 of Schwarze, shows no uridine incorporation into particles of 50 S and of 30 S, but some very weak labelling of the 80 S cytoplasmic monosomes.Nitrogen deficient Chlorella cells also incorporate uridine mainly into the 50 S and 30 S particles. When chase treatment with unlabelled uridine is performed under recovering conditions, the label shifts into the 70 S particles as well as into the 80 S cytoplasmic ribosomes.The results indicate that in Chlorella, uridine is incorporated into chloroplast ribosome precursors rather than into particles of nuclear origin.