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1.
A comparison of the unfolding and dissociation of the large ribosome subunits from Rhodopseudomonas·spheroides N.C.I.B. 8253 and Escherichia coli M.R.E. 600 总被引:1,自引:1,他引:0
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1. The behaviour of the large ribosomal subunit from Rhodopseudomonas spheroides (45S) has been compared with the 50S ribosome from Escherichia coli M.R.E. 600 (and E. coli M.R.E. 162) during unfolding by removal of Mg(2+) and detachment of ribosomal proteins by high univalent cation concentrations. The extent to which these processes are reversible with these ribosomes has also been examined. 2. The R. spheroides 45S ribosome unfolds relatively slowly but then gives rise directly to two ribonucleoprotein particles (16.6S and 13.7S); the former contains the intact primary structure of the 16.25S rRNA species and the latter the 15.00S rRNA species of the original ribosome. No detectable protein loss occurs during unfolding. The E. coli ribosome unfolds via a series of discrete intermediates to a single, unfolded ribonucleoprotein unit (19.1S) containing the 23S rRNA and all the protein of the original ribosome. 3. The two unfolded R. spheroides ribonucleoproteins did not recombine when the original conditions were restored but each simply assumed a more compact configuration. Similar treatments reversed the unfolding of the E. coli 50S ribosomes; replacement of Mg(2+) caused the refolding of the initial products of unfolding and in the presence of Ni(2+) the completely unfolded species (19.1S) again sedimented at the same rate as the original ribosomes (44S). 4. Ribosomal proteins (25%) were dissociated from R. spheroides 45S ribosomes by dialysis against a solution with a Na(+)/Mg(2+) ratio of 250:1. During this process two core particles were formed (21.2S and 14.2S) and the primary structures of the two original rRNA species were conserved. This dissociation was not reversed. With E. coli 50S approximately 15% of the original ribosomal protein was dissociated, a single 37.6S core particle was formed, the 23S rRNA remained intact and the ribosomal proteins would reassociate with the core particle to give a 50S ribosome. 5. The ribonuclease activities in R. spheroides 45S and E. coli M.R.E. 600 and E. coli M.R.E. 162 50S ribosomes are compared. 6. The observations concerning unfolding and dissociation are consistent with previous reports showing the unusual rRNA complement of the mature R. spheroides 45S ribosome and show the dependence of these events upon the rRNA and the importance of protein-protein interactions in the structure of the R. spheroides ribosome. 相似文献
2.
The soluble fraction extracted from Escherichia coli A19 (Hfr, rel met rns) during early and late times of phenotypic and genotypic induced relaxed control have been examined for the possible accumulation of ribosomal proteins (r-proteins) and rRNA species during this time of unbalanced macromolecular synthesis. Ribosomal proteins and rRNA species were not found to accumulate within the soluble fraction at any time during this period of relaxed control; even after the typical rRNA accumulation had ceased, r-proteins did not accumulate. It is concluded, from these and related observations, that the r-proteins and rRNA species known to be produced during relaxation must immediately associate to form the unusual ribonucleoprotein particles (e.g. 'relaxed particles' and 'chloramphenicol particles') characteristic of periods of relaxed control. Since r-proteins do not accumulate even when net RNA accumulation halts, it appears that some elements of the normal, basic co-ordination between rRNA and r-protein synthesis/stability persist even during relaxed control. 相似文献
3.
The effect of polyamines on in vitro reconstitution of Escherichia coli 30S and 50S ribosomal subunits has been studied. Spermidine stimulated the reconstitution of 30S particles from 16S rRNA lacking the methyl groups on two neighboring adenines and total proteins of 30S subunits at least 1.6-fold. The reconstitution of 30S particles from normal 16S rRNA and total proteins of 30S subunits exhibited only slight spermidine stimulation. However, the optimal Mg2+ concentration of the reconstitution was decreased from 20 mM to 16 mM in the presence of 3 mM spermidine. In the absence of spermidine the assembly of 30S particles from normal 16S rRNA was more rapid than the assembly from 16S rRNA lacking the methyl groups on two neighboring adenines. The reconstitution of 50S particles from 23S and 5S rRNA and total proteins of 50S subunits was not influenced greatly by spermidine. Gel electrophoresis results, from reconstitution experiments of 30S particles from 16S rRNA lacking the methyl groups on two neighboring adenines and total proteins of 30S subunits, showed that the assembly of S1 and S9 proteins to 23S core particles was stimulated by spermidine during reconstitution. The relationship of polyamine effects on in vitro ribosome assembly from its constituents to in vivo ribosome assembly is discussed. The reconstitution of Bacillus subtilis 30S particles from 16S rRNA and total proteins of 30S subunits was also stimulated approximately 1.3-fold by 3 mM spermidine. 相似文献
4.
Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly. 相似文献
5.
The effect of magnesium starvation upon the fate of individual ribosomal proteins was studied in Escherichia coli. During a 21 h incubation in the absence of Mg2+ the 30 S subunit was more susceptible to degradation, retaining an average 31.9% of its ribosomal proteins as compared to 40.0% for the 50 S subunit. An examination of those 50-S proteins dissociated to a lesser extent than the average value (L1, L2, L3, L7, L10, L13, L16, L17, L19, L21, L22, L23, and L29) revealed that, with the exception of L16, all were classified by Dohme and Nierhaus [5] as tightly bound. Of the ribosomal proteins dissocated during magnesium starvation only five were reincorporated (and these to a minimal degree) during recovery of cells in a medium containing Mg2+. These studies suggest that ribosomal proteins once released from the ribosome particles during magnesium starvation are not reutilized in the assembly of new subunits. 相似文献
6.
Earlier studies have shown that the reconstitution of Escherichia coli 50S as well as 30S ribosomal subunits from component rRNA and ribosomal protein (r-protein) molecules in vitro is not completely cooperative and binding of more than one r-protein to a single 16S rRNA (or 23S rRNA) molecule is required to initiate a successful 30S (or 50S) ribosome assembly reaction. We first confirmed this conclusion by carrying out 30S subunit reconstitution in the presence of a constant amount of 16S rRNA together with various amounts of total 30S r-proteins (TP30) and by analyzing the physical state of reconstituted particles rather than by assaying protein synthesizing activity of the particles as was done in the earlier studies. As expected, under conditions of excess rRNA, the efficiency of 30S subunit reconstitution per unit amount of TP30 decreased greatly with the decrease in the ratio of TP30 to rRNA, indicating the lack of complete cooperativity in the assembly reaction. We then asked the question whether the cooperativity of ribosome assembly is complete in vivo. We treated exponentially growing E coli cells with low concentrations of chloramphenicol which is known to inhibit protein synthesis without inhibiting rRNA synthesis, creating conditions of excess synthesis of rRNA relative to r-proteins. Several concentrations of chloramphenicol (ranging from 0.4 to 4.0 micrograms/ml) were used so that inhibition of protein synthesis ranged from 40 to 95%. Under these conditions, we examined the synthesis of RNA, ribosomal proteins and 50S ribosomal subunits as well as the synthesis of total protein. We found that the synthesis of 50S subunits was not inhibited as much as the synthesis of total protein at lower concentrations of chloramphenicol, but the degree of inhibition of 50S subunit synthesis increased sharply with increasing concentrations of chloramphenicol and was in fact greater than the degree of inhibition of total protein synthesis at chloramphenicol concentrations of 2 micrograms/ml or higher. The inhibition of 50S subunit synthesis was significantly greater than the inhibition of r-protein synthesis at all chloramphenicol concentrations examined. These data are consistent with the hypothesis that the cooperativity of ribosome assembly in vivo is also not complete as is the case for in vitro ribosome reconstitution, but are difficult, if not impossible, to explain on the basis of the complete cooperativity model.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
7.
The metabolism of the ribosomal and soluble protein components of Aerobacter aerogenes was examined during its incubation in a Mg(++)-deficient medium. Bacteria were exposed to leucine-H(3) during the exponential growth period preceding Mg(++) starvation, and extracts were prepared after intervals of starvation and were centrifuged through gradients of sucrose to separate ribosomal from soluble proteins. Ribosomal proteins synthesized during the preceding exponential growth were slowly lost from the ribosomes; after 8 hr of starvation, few, if any, sedimented with ribosomes. Losses of total protein, together with the known rate of ribosome decay during Mg(++) starvation, suggested that these ribosomal proteins are ultimately degraded to acid-soluble products and account for all protein lost by the starving cells. These conclusions were supported by studies of Mg(++) starvation in a uracil-requiring strain of A. aerogenes: during uracil starvation a smaller fraction of the proteins synthesized were ribosomal, and the fraction of protein which subsequently decayed during Mg(++) starvation was correspondingly less. During recovery from Mg(++) starvation, proteins, lost from disintegrated ribosomes, were not detectably reutilized into new particles even before their degradation to acid-soluble products was complete. Synthesis of soluble proteins continued for more than 24 hr of starvation at a rate per milliliter close to 45% of the instantaneous rate per milliliter of the exponentially growing bacteria at the time Mg(++) was removed. This value agreed with that found previously for synthetic rates of deoxyribonucleic acid, transfer ribonucleic acid, and ribosomal ribonucleic acid during starvation relative to rates during exponential growth. 相似文献
8.
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. 相似文献
9.
《Journal of molecular biology》1996,263(3):396-410
The ability of mutant 23 S ribosomal RNA to form particles with proteins of the large ribosomal subunitin vivowas studied. A series of overlapping deletions covering the entire 23 S rRNA, were constructed in the plasmid copy of anE. coli23 S rRNA gene. The mutant genes were expressedin vivousing an inducibletacpromoter. Mutant species of 23 S rRNA, containing deletions between positions 40 and 2773, were incorporated into stable ribonucleoprotein particles. In contrast, if one end of the 23 S rRNA was deleted, the mutant rRNA was unstable and did not form ribosomal particles. Protein composition of the mutant particles was specific; the presence of the primary rRNA-binding proteins corresponded to their known binding sites. Furthermore, several previously unknown ribosomal protein binding sites in 23 S rRNA were identified. Implications of the results on ribosome assembly are discussed. 相似文献
10.
Halobacterium cutirubrum ribosomes. Properties of the ribosomal proteins and ribonucleic acid 总被引:4,自引:0,他引:4
L. P. Visentin C. Chow A. T. Matheson M. Yaguchi F. Rollin 《The Biochemical journal》1972,130(1):103-110
1. The 30S ribosomal subunit of the extreme halophile Halobacterium cutirubrum is unstable and loses 75% of its ribosomal protein when the 70S ribosome is dissociated into the two subunits. A stable 30S subunit is obtained if the dissociation of the 70S particle is carried out in the presence of the soluble fraction. 2. A fractionation procedure was developed for the selective removal of groups of proteins from the 30S and 50S subunits. When the ribosomes, which are stable in 4m-K(+) and 0.1m-Mg(2+), were extracted with low-ionic-strength buffer 75-80% of the 30S proteins and 60-65% of the 50S proteins as well as the 5S rRNA were released. The proteins in this fraction are the most acidic of the H. cutirubrum ribosomal proteins. Further extraction with Li(+)-EDTA releases additional protein, leaving a core particle containing either 16S rRNA or 23S rRNA and about 5% of the total ribosomal protein. The amino acid composition, mobility on polyacrylamide gels at pH4.5 and 8.7, and the molecular-weight distribution of the various protein fractions were determined. 3. The s values of the rRNA are 5S, 16S and 23S. The C+G contents of the 16S and 23S rRNA were 56.1 and 58.8% respectively and these are higher than C+G contents of the corresponding Escherichia coli rRNA (53.8 and 54.1%). 相似文献
11.
Ribosome subunit assembly in bacteria is a fast and efficient process. Among the nonribosomal proteins involved in ribosome biogenesis are RNA helicases. We describe ribosome biogenesis in Escherichia coli strains lacking RNA helicase DeaD (CsdA) or DbpA. Ribosome large subunit assembly intermediate particles (40S) accumulate at 25 degrees C and at 37 degrees C in the absence of DeaD but not without DbpA. 23S rRNA is incompletely processed in the 40S and 50S particles of the DeaD(-) strain. Pulse labeling showed that the 40S particles are converted nearly completely into functional ribosomes. The rate of large ribosomal subunit assembly was reduced about four times in DeaD-deficient cells. Functional activity tests of the ribosomal particles demonstrated that the final step of 50S assembly, the activation step, was affected when DeaD was not present. The results are compatible with the model that predicts multiple DeaD-catalyzed structural transitions of the ribosome large subunit assembly. 相似文献
12.
Escherichia coli 3'-terminal 16S rRNA sequence modulated fidelity during translation 总被引:2,自引:0,他引:2
The ribosome is a central component of the protein synthetic apparatus. Although progress has been made in characterizing the functional role of many of the ribosomal proteins, the properties of ribosomal RNA and its role in ribosome structure and function are not well understood. To investigate the working properties of the highly conserved 3'-end of 16S rRNA, a site-specific deletion was made directly within the 16S rRNA molecule. The terminal deletion did not impair in vitro 30S subunit assembly, but the particles produced lost translational fidelity in an in vitro translation system primed with natural mRNA. 相似文献
13.
Identification of novel Escherichia coli ribosome-associated proteins using isobaric tags and multidimensional protein identification techniques
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Jiang M Sullivan SM Walker AK Strahler JR Andrews PC Maddock JR 《Journal of bacteriology》2007,189(9):3434-3444
Biogenesis of the large ribosomal subunit requires the coordinate assembly of two rRNAs and 33 ribosomal proteins. In vivo, additional ribosome assembly factors, such as helicases, GTPases, pseudouridine synthetases, and methyltransferases, are also critical for ribosome assembly. To identify novel ribosome-associated proteins, we used a proteomic approach (isotope tagging for relative and absolute quantitation) that allows for semiquantitation of proteins from complex protein mixtures. Ribosomal subunits were separated by sucrose density centrifugation, and the relevant fractions were pooled and analyzed. The utility and reproducibility of the technique were validated via a double duplex labeling method. Next, we examined proteins from 30S, 50S, and translating ribosomes isolated at both 16 degrees C and 37 degrees C. We show that the use of isobaric tags to quantify proteins from these particles is an excellent predictor of the particles with which the proteins associate. Moreover, in addition to bona fide ribosomal proteins, additional proteins that comigrated with different ribosomal particles were detected, including both known ribosomal assembly factors and unknown proteins. The ribosome association of several of these proteins, as well as others predicted to be associated with ribosomes, was verified by immunoblotting. Curiously, deletion mutants for the majority of these ribosome-associated proteins had little effect on cell growth or on the polyribosome profiles. 相似文献
14.
15.
The pool size of free individual ribosomal proteins present in the cell sap of Escherichia coli has been determined by pulse-labelling a culture before a chase with cold marker.Ribosomes plus ribosomal precursor particles were prepared together and the proteins from this fraction purified. The specific radioactivity of each 30 S and 50 S protein was measured at the time of pulse and at the various times of chase: unequal labelling was already observed at the time of pulse; the kinetics of chase of most 30 S proteins reached a plateau very rapidly; the kinetics of 50 S proteins were more variable. Precise calculation of individual pool size was carried out using the mathematical model described in the Appendix. Almost all ribosomal 30 S proteins have a pool size close to zero. Only four 30 S proteins (S10, S16, S17 and S18) have a sizeable pool (2 to 6% of the corresponding ribosomal protein). Most 50 S proteins have a small pool size (1 to 2%). The free ribosomal proteins of the pool are transferred to mature ribosomes; the half-life of these proteins in the pool has been calculated (0 to 1·4 min). Finally, as judged from the kinetic data, no degradation of ribosome-bound protein was apparent. The significance of the results is discussed with respect to the function of ribosome and the process of ribosome biogenesis. 相似文献
16.
Precursor Ribosomal Ribonucleic Acid and Ribosome Accumulation In Vivo During the Recovery of Salmonella typhimurium from Thermal Injury 总被引:11,自引:2,他引:9
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When cells of S. typhimurium were heated at 48 C for 30 min in phosphate buffer (pH 6.0), they became sensitive to Levine Eosin Methylene Blue Agar containing 2% NaCl (EMB-NaCl). The inoculation of injured cells into fresh growth medium supported the return of their normal tolerance to EMB-NaCl within 6 hr. The fractionation of ribosomal ribonucleic acid (rRNA) from unheated and heat-injured cells by polyacrylamide gel electrophoresis demonstrated that after injury the 16S RNA species was totally degraded and the 23S RNA was partially degraded. Sucrose gradient analysis demonstrated that after injury the 30S ribosomal subunit was totally destroyed and the sedimentation coefficient of the 50S particle was decreased to 47S. During the recovery of cells from thermal injury, four species of rRNA accumulated which were demonstrated to have the following sedimentation coefficients: 16, 17, 23, and 24S. Under identical recovery conditions, 22, 26, and 28S precursors of the 30S ribosomal subunit and 31 and 48S precursors of the 50S ribosomal subunit accumulated along with both the 30 and 50S mature particles. The addition of chloramphenicol to the recovery medium inhibited both the maturation of 17S RNA and the production of mature 30S ribosomal subunits, but permitted the accumulation of a single 22S precursor particle. Chloramphenicol did not affect either the maturation of 24S RNA or the mechanism of formation of 50S ribosomal subunits during recovery. Very little old ribosomal protein was associated with the new rRNA synthesized during recovery. New ribosomal proteins were synthesized during recovery and they were found associated with the new rRNA in ribosomal particles. The rate-limiting step in the recovery of S. typhimurium from thermal injury was in the maturation of the newly synthesized rRNA. 相似文献
17.
Identification of a Precursor Pool of Ribosome Protein in Escherichia coli 总被引:3,自引:0,他引:3
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Melvin Santer Trenton K. Ruebush John Van Brunt Eben Oldmixon Russell Hess Paul Primakoff Philip Palade 《Journal of bacteriology》1968,95(4):1355-1367
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. 相似文献
18.
Jiang M Datta K Walker A Strahler J Bagamasbad P Andrews PC Maddock JR 《Journal of bacteriology》2006,188(19):6757-6770
The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which is poorly understood. Although several bona fide assembly factors have been identified, their precise functions and temporal relationships are not clearly defined. Here we describe the involvement of an Escherichia coli GTPase, CgtA(E), in late steps of large ribosomal subunit biogenesis. CgtA(E) belongs to the Obg/CgtA GTPase subfamily, whose highly conserved members are predominantly involved in ribosome function. Mutations in CgtA(E) cause both polysome and rRNA processing defects; small- and large-subunit precursor rRNAs accumulate in a cgtA(E) mutant. In this study we apply a new semiquantitative proteomic approach to show that CgtA(E) is required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribosomal subunit. Moreover, we demonstrate the interaction with the 50S ribosomal subunits of specific nonribosomal proteins (including heretofore uncharacterized proteins) and define possible temporal relationships between these proteins and CgtA(E). We also show that purified CgtA(E) associates with purified ribosomal particles in the GTP-bound form. Finally, CgtA(E) cofractionates with the mature 50S but not with intermediate particles accumulated in other large ribosome assembly mutants. 相似文献
19.
Atomic resolution structures of 50S and 30S ribosomal particles have recently been solved by X-ray diffraction. These ribosomal structures show often unusual folds of ribosomal RNAs and proteins, and provide molecular explanations for fundamental aspects of translation. In the 50S structure, the active site for peptide bond formation was localized and found to consist of RNA. The ribosome is thus a ribozyme. In the 30S structures, tRNA binding sites were located, and molecular mechanisms for ribosomal fidelity were proposed. The 30S subunit particle has three globular domains, and relative movements of these domains may be required for translocation of the ribosome during protein synthesis. The structures are consistent with and rationalize decades of biochemical analysis of translation and usher in a molecular age in understanding the ribosome. 相似文献
20.
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. 相似文献