Association of 5S ribonucleic acid to 50S ribosomal subunits of Escherichia coli and Bacillus subtilis

Morell, Pierre; Marmur, Julius

doi:10.1021/bi00843a035

Cited by 44 publications

(5 citation statements)

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“…The initial partially unfolded intermediates from E. coli were readily refolded to the original 50S species whereas the completely unfolded 19.1 S species was not. Difficulty in reversing the complete E. coli unfolding sequence has been observed previously (Gavrilova et al, 1966;Atsmon et al, 1967;Morell & Marmur, 1968;Roberts & Walker, 1970). The apparent reversal with Ni2+ present, although in agreement with the observation by Tal (1969), should be viewed with caution in view of the tendency of this ion to increase the sedimentation coefficient of all species present.…”

Section: Discussionsupporting

confidence: 86%

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

Robinson

Sykes

1973

Biochemical Journal

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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.

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Section: Discussionsupporting

confidence: 86%

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

Robinson

Sykes

1973

Biochemical Journal

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“…The first stage of the unfolding seems to be reversible, i.e., the particles can spontaneously recover their initial compactness and biological activity upon the restoration of the Mg*+ content [2 1,96, 97,100]. Probably this does not occur if the 50 S (or 60 S) subparticle loses its 5 S RNA [99,107,108]. The final stage of unfolding may not reverse to biologically active particles upon the restoration of the Mg*+ content [96][97][98]125,126] ; however, the recovery of the initial structure and biological activity can be attained in this case if the particles are subjected to heat activation [125,126].…”

Section: Discoverymentioning

confidence: 99%

“…It should be noted that in the process of disassembly the large ribosomal subparticle loses not only the proteins, but its 5 S RNA as well. The splitting of 5 S RNA proceeds simultaneously with the splitting off of a definite group of proteins; it has been established that on disassembly of E. koli ribosomes, the 43 S-40 S particles (and perhaps the 38 S-36 S particles as well) still contain 5 S RNA, while the 28 S-25 S particles are already completely devoid of it [99,148,1641. Stepwise stripping of proteins with high salt concentrations was also achieved with some animal ribosomes [156-1601. …”

Section: Discoverymentioning

confidence: 99%

Structural transformations of ribosomes (dissociation, unfolding and disassembly)

Spirin

1974

FEBS Letters

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“…Its release results in loss of biological activity of the large subunit (8) and apparently requires different conditions in prokaryotic and eukaryotic ribosomes. In 70S particles, either magnesium depletion by chelating agents (9) or competition for magnesium by high concentrations of monovalent ions released 5S RNA (9,10), whereas in 80S particles, magnesium chelation by EDTA was found to have widely differing effects in different cells: it released 5S RNA from fungal ribosomes (11) and rat liver ribosomes (12), but not from ribosomes of HeLa cells (13) or rabbit reticulocytes (14). In rabbit reticulocytes, EDTA was effective only at pH 9.5. In this paper it is shown that 5S RNA is released from mammalian ribosomes by either EDTA or high concentrations of monovalent ions.…”

mentioning

confidence: 99%

Isolation of a 5S RNA-Protein Complex from Mammalian Ribosomes

Blobel

1971

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

116

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(14). In rabbit reticulocytes, EDTA was effective only at pH 9.5. In this paper it is shown that 5S RNA is released from mammalian ribosomes by either EDTA or high concentrations of monovalent ions. The species released is not a naked molecule but a ribonucleoprotein, in which the 5S RNA is associated with a single protein. METHODS The procedures for the preparation of free ribosomes from rat liver and rabbit reticulocytes, the dissociation of ribosomes into biologically active subunits, and the subsequent separation of these subunits in sucrose gradients have been described (15,16). Gradient fractions were collected, made 10 mM in magnesium, and treated with 2 volumes of ethanol at -20°C. The resultant precipitate was recovered after 20 hr by centrifugation for 20 min at 2000 X g. Electrophoresis was performed in a vertical electrophoresis cell (E -C Apparatus Corp., Philadelphia, Pa.) in slabs 3-mm thick, each provided with eight slots.For the electrophoresis of 4S and 5S RNA, the procedure of Peacock and Dingman (17) was slightly modified as follows: a 5% acrylamide gel without agarose was used, and EDTA 1881 was omitted from both the gel and the buffer; instead, both contained 50 mM KCl and 2 mM MgCl2. The alcohol precipitates of the gradient fractions were dissolved in 15% sucrose, and 25-Al aliquots were layered into the slots. Electrophoresis was performed at 15°C for 4 hr at a constant current of 100 mA and at the end of the procedure the gels were stained with "Stains-all" (18).Ribosomal proteins were electrophoresed in sodium dodecyl sulfate gels by the procedure described by Maizel (19) in the E -C apparatus. After electrophoresis, the gels were stained in Coomassie Brilliant Blue. RESULTSDuring a study on the dissociation of mammalian ribosomes into biologically active subunits in high concentrations of monovalent ions in the presence of various magnesium concentrations, I noticed that below a critical divalent-cation concentration both ribosomal subunits were converted into slower-sedimenting, distinct derivatives (sharp symmetrical peaks). Table 1 summarizes the changes in sedimentation rates of both ribosomal subunits, when sedimented into sucrose gradients containing 500 mMI KCl and magnesium concentrations varying from 5.0 to 0.0 mM. Below 2.0 mM magnesium, the large ribosomal subunit (LO) forms a derivative (L') with a greatly reduced sedimentation rate, while the small subunit (SO) undergoes two distinct changes: a derivative (S1) that sediments slightly more slowly is formed at concentrations <2 mM magnesium and a derivative (S2) that sediments much more slowly appears below 0.5 mM magnesium. Ribosomal subunits, dissociated with EDTA (20), sediment with rates identical to L1 and S2. The derivatives of both ribosomal subunits are biologically inactive, i.e., they do not associate and do not synthesize polyphenylalanine in the presence of poly(U), whereas LO and S are active (15).The EDTA-induced conversion of the large ribosomal subunit from a 60S to a 47S form noted by Tashiro and Siekev...

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Association of 5S ribonucleic acid to 50S ribosomal subunits of Escherichia coli and Bacillus subtilis

Cited by 44 publications

References 28 publications

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

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

Structural transformations of ribosomes (dissociation, unfolding and disassembly)

Isolation of a 5S RNA-Protein Complex from Mammalian Ribosomes

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