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Treatment of Escherichia coli ribosomes with the protein reagent 2,3-dimethylmaleic anhydride is accompanied by inactivation of polypeptide polymerization and by dissociation of ribosomal proteins. Regeneration of the modified amino groups at pH 6.0 is followed by reactivation and reconstitution of the ribosomes. Prior to regeneration of the amino groups, ribosomal particles and split proteins can be separated by centrifugation, which allows the preparation of new protein-deficient particles. The ribosomal particles obtained by three successive treatments with 2,3-dimethyl-maleic anhydride at a molar ratio of reagent to ribosome equal to 16,000 lack proteins S1, S2, S3, S5, S10, S13, S14, L7, L8, L10, L11, L12, and L20 and have lost part of proteins S4, L1, L6, L16, and L25. This new procedure to obtain protein-deficient ribosomal particles is mild and might be useful to dissociate other protein-containing structures in addition to ribosomes.  相似文献   

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Summary The exchange of ribosomal proteins among ribosomes of E. coli has been measured, using a density label technique. As expected most of the proteins do not exhange appreciably. However a substantial fraction of each of proteins S1, S2, S21, L7/L12, L9, L10, L11, L26 and L33 is found to exchange, but exchange of S1, S2, L7/L12, L10, L11 and L26 is found to occur in vitro after lysis of the cells, and therefore it is not possible to say whether or not these proteins also exchange in vivo. In contrast S21, L9 and L33 do not exchange after lysis of the cells and we therefore conclude that these proteins exchange in vivo. The maximum level of exchange of S21, L9 and L33 is attained so rapidly that we were unable to show whether or not it was dependent on protein synthesis.  相似文献   

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Biogenesis of ribosomes: free ribosomal protein pools in Escherichia coli   总被引:3,自引:0,他引:3  
Proteins from ribosomal subunits (30 s and 50 s) have been fractionated into split (SP-30 and SP-50) and core (core-30 and eore-50) proteins. Antisera prepared in rabbit against them are shown to be highly group-specific as judged by the Ouchterlony (1967) double diffusion test and by precipitin reaction in solution. Various parameters which influence the immuno-precipitation of these proteins by specific antisera have been investigated. It is demonstrated that under controlled conditions this provides a sensitive and reliable method for characterization and quantitative estimation of free ribosomal proteins. The technique has been successfully applied in the investigations of various properties of free ribosomal protein pools existing in Escherichia coli. It is concluded that free ribosomal proteins in E. coli may constitute 8 to 14% of total soluble proteins under different growth conditions. Relative pool sizes of four classes of proteins expressed as a percentage of the total soluble proteins in bacteria grown in L broth (doubling period, 30 min) are estimated to be core-30, 2.1; core-50, 3.4; SP-30, 3.6; SP-50, 5.0. From the studies on bacteria grown in different media (doubling period, 30, 42 and 60 min), we further conclude that the amount of free proteins increases with the growth rate so as to constitute a constant fraction (7 to 9%) of total ribosomal proteins. Relative pool-sizes corresponding to four classes of ribosomal proteins, however, remain unaltered by different growth rate.  相似文献   

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Magnesium binding by Escherichia coli ribosomes   总被引:12,自引:0,他引:12  
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We have used a series of N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) maleimide spin labels of different length to label, covalently and selectively, the most reactive sulfhydryl groups of 70S ribosomal proteins of Escherichia coli. Under short periods of labeling (1--2 min), less than two spin labels per ribosome are incorporated and were shown to be distributed mainly on five ribosomal proteins in the following order: S18 greater than S21, L27 greater than S17, and S12. With a long period of labeling (3 h) up to 13 spin labels are attached to the ribosome, and protein S1 is the most labeled. The shape of the electron paramagnetic resonance (epr) signal shows two components with a predominance for the strongly immobilized orientation, and the percentage of these components in each spectra has been evaluated. When the distance between the nitroxide group and the maleimide-attaching group exceeds 6 A (1 A = 0.1 nm) the strongly immobilized orientation disappears. The effect of magnesium ions on these selectively spinlabeled ribosomes shows that the dissociation into subunits does not affect the epr signal, but more spin labels are incorporated into the subunits if labeling is performed under conditions of dissociation.  相似文献   

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The dissociation of purified 70 S.E. coli ribosomes, induced by the dissociation factor DF, has been studied by submitting the reaction mixtures to electrophoresis on polyacrylamide gels. The electrophoretic analysis of the ribosome mixtures revealed a heterogeneity which escaped detection by conventional sucrose gradient centrifugation. Increasing amounts of DF in the reaction mixtures converted 70 S ribosomes to particles (designated 70 S (I)) which migrate slower in the electric field than the original 70 S ribosomes. These 70 S (I) ribosomes still consist of both subunits. They dissociate upon further raising the DF concentration.  相似文献   

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Crystallization of Escherichia coli ribosomes   总被引:1,自引:0,他引:1  
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Exposure of cells of Escherichia coli to mitomycin C (5 mug/ml) resulted in a marked change in the sedimentation profiles of the cell-free extracts, indicating a specific decomposition of ribosomal particles. When the extracts were prepared in the presence of 0.01 m Mg(++) and analyzed by sucrose density gradient centrifugations, the 100S fraction disappeared rapidly from the treated cells. The 70S ribosomes were also degraded, but more slowly, with a concomitant accumulation of a fraction having a sedimentation coefficient of about 50S. However, decomposition of the 70S ribosomes was preceded by an almost complete loss of the 50S ribosomal subunits, as revealed by sedimentation analyses in the presence of 10(-4)m Mg(++). Synthesis of the ribosomes in the treated cells was also suppressed, being demonstrated by a lower incorporation of uracil-2-(14)C into the ribosomal fractions. However, the change in the ribosomal profile in the treated cells apparently resulted from the decomposition of pre-existing ribosomes, rather than from the inhibition of the net synthesis of ribosomes. Sedimentation analyses and chromatography of the nucleic acids extracted from the treated cells indicated extensive but delayed degradation of the ribosomal ribonucleic acid (RNA), but not of the soluble RNA or deoxyribonucleic acid fractions. Altered structure of the ribosomes in the treated cells was also indicated by their lower melting temperature, broadened thermal profile, higher electrophoretic mobility, and extreme sensitivity to ribonuclease treatment, compared with normal ribosomes. The synthesis of messenger RNA was inhibited progressively with time in the treated cells.  相似文献   

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