Localization of ribosome precursors in Escherichia coli

Girolamo, M. Di; Hinckley, E.; Busiello, Enzo

doi:10.1016/0005-2787(68)90047-6

Cited by 10 publications

(3 citation statements)

References 12 publications

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“…Consistent with the fact that ribosomal RNA precursors first appear on a rapidly sedimenting cell fraction thought to be membrane are the observations that precursors to ribosomal subunits are found on total sediment (Mg++ sediment and sediment) (1) and, more specifically, that ribosomal subunit precursors can be found on membrane isolated in low Mg++ (9). The most likely explanation for these observations is that the ribosomal RNA precursor is synthesized on cell membrane.…”

Section: Discussionsupporting

confidence: 64%

Cellular Site of Escherichia coli Ribosomal RNA Synthesis

Haywood

1971

Proc. Natl. Acad. Sci. U.S.A.

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Escherichia coli, grown for a generation after the addition of ['4C]uracil and for one minute after a pulse of [3H]uracil, were lysed in the presence of 6 mM Mg++, and divided into a 30,000 X g "supernatant" and sediment. The sediment was resuspended in buffer containing no divalent cations, and again centrifuged to give the "Mg++ sediment" and the remaining "sediment". MATERIALS AND METHODSBacteria and growth medium were described (1).Buffers. TKA is 10 mM Tris (pH8)-40 mM KCl-10 mM NaN3. TMKA is TKA plus 6 mM MgCl2. TMKAS is TMKA plus 40 mM Na2SO4.Detergents. The following detergents were used: sodium deoxycholate, sodium dodecyl sulfate, Sarkosyl NL 97 (sodium lauryl sarcosinate), Triton X-200 (sodium alkylaryl polyether sulfonate), Triton X-400 (stearyl-dimethylbenzylammonium chloride), Triton X-100 (alkylaryl polyether alcohol), Brij 58 (polyoxyethylenecetyl ether), Nonidet P-40 (p-octylphenol ethoxylate), Tween 80 (polyoxyethylene sorbitan monooleate), and digitonin.Lysis and Fractionation of Lysate. Harvesting and lysis of the cells by the lysozyme freeze-thaw method was previously described (1). In brief, cells were chilled, centrifuged, and resuspended in buffer at a concentration of at least 2 X 1010 cells/ml. Lysozyme, 1 mg/ml, was added, and after 3 min the cells were frozen and thawed twice. DNase, 10 ug/ml, was added; 10 min after the addition of DNase, the lysate was centrifuged at 13,000 X g for 10 min. The sediment was re-435 suspended in 0.01 M Tris (pH 8)-6 mM magnesium acetate or in TMKA (1.5 times the volume of the original lysate), and centrifuged at 30,000 X g for 15 min. This step was repeated and the supernatants were combined to give the "supernatant."The sediment was then resuspended in 0.01 M Tris (pH 8) or TKA and centrifuged at 30,000 X g for 15 min. This step was repeated twice and the supernatants combined. This yields the RNA-containing structures that are bound to the sediment in the presence of Mg++ and are called "Mg++ sediment." In some experiments, if the glass centrifuge tube was viewed against a black background, a faint opalescence could be seen near the bottom of the tube. In this case, the supernatant and Mg++ sediment were recentrifuged at 30,000 X g for 30 min and the upper 2/3 of the solution was used for RNA extraction.The remaining sediment was resuspended in 0.01 M Tris (pH 8) to give the "sediment." In some cases, the sediment was layered on successive layers of 8 ml of 40% (w/v) sucrose, 16 ml of 50% sucrose, and 12 ml of 70% sucrose, and centrifuged overnight at 23,000 rpm in the Spinco SW-27 rotor at 50C. The interface between 50 and 70% sucrose was collected.Extraction of RNA. After l/lo volume of 5% sodium dodecyl sulfate was added to the sample, the RNA was extracted by the method of Kirby (2).Electrophoresis. The method of Summers (3) was used to prepare 2.7% acrylamide-0.5% agarose gels. The 4.1% gels were prepared by the method of Loening (4). Buffer G (3) was used in both cases. Electrophoresis was at room temperature at a constant current of 5...

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Section: Discussionsupporting

confidence: 64%

Cellular Site of Escherichia coli Ribosomal RNA Synthesis

Haywood

1971

Proc. Natl. Acad. Sci. U.S.A.

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“…We previously demonstrated that the sedimentation behavior of ribosomal precursor subunits depends on the method used for cell extract preparation (1) and that those precursor subunits that cosediment with mature subunits can be converted to as many as five more slowly sedimenting particles similar to those observed in other stud- ies of ribosomal subunit assembly (1,2,13,14,22). Although membrane fractions have been shown to contain both ribosomal precursor subunits (4,18) and precursor ribosomal RNA (8), most other studies of ribosomal subunit assembly have used methods of cell disruption involving detergent treatments or high shear forces. These methods would disrupt membrane fragments that might be associated with precursor ribosomal subunits.…”

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confidence: 76%

Effects of Detergents on Ribosomal Precursor Subunits of Bacillus megaterium

Body

Brownstein

1978

J Bacteriol

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Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.

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“…The fractions prepared in this study cannot be exactly related to those used in all other studies in which membrane RNA and ribosomes have been reported. In some studies (7,12), the deoxyribonuclease was added before detergents. All spheroplasts are probably not equally affected by osmotic shock or detergents.…”

Section: Discussionmentioning

confidence: 99%

“Compartmentalization” of Escherichia coli Ribosomes and Ribonucleic Acid

Varricchio

1972

J Bacteriol

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Escherichia coli spheroplasts lysed by Brij 58 and deoxycholate were separated into supernatant (S) and membrane fractions by low-speed centrifugation. The membrane fraction was further divided into that which was releasable by deoxyribonuclease (fraction D) and that which was not (M). In the presence of 10-2 M Mg2+, the S, D, and M fractions contained, respectively, 60, 20, and 20% of the total cellular ribonucleic acid (RNA). Ribosomal and transfer RNA (rRNA, tRNA) were found in each fraction. The M + D fraction RNA was labeled more by a pulse label. Incorporation of uracil into the D fraction continued only as long as the uptake of exogenous uracil, suggesting that this was a major primary site of RNA synthesis. From pulse-labeled cells, each fraction contained precursor rRNA, and there was a 10S RNA in the M fraction. Ninety per cent of the ribosomal subunits and the ribosomal precursor particles, 26 and 43S, were in the S fraction. Precursor RNA (17S) was found in the 26S precursor particles. The D fraction contained 38% of the polysomes (this does not consider polysomes, if any, of the M fraction) which were labeled four times as much as the supernatant polysomes by a 1-min pulse of uracil. These results are interpreted to mean that new RNA is associated with a cytoplasmic membrane-RNA polymerase-DNA complex.

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Localization of ribosome precursors in Escherichia coli

Cited by 10 publications

References 12 publications

Cellular Site of Escherichia coli Ribosomal RNA Synthesis

Cellular Site of Escherichia coli Ribosomal RNA Synthesis

Effects of Detergents on Ribosomal Precursor Subunits of Bacillus megaterium

“Compartmentalization” of Escherichia coli Ribosomes and Ribonucleic Acid

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