Size heterogeneity of the large ribosomal subunits and conservation of the small subunits in eucaryote evolution

Cammarano, Piero; Romeo, Antonino; Gentile, Marco; Felsani, Armando; Gualerzi, Claudio O.

doi:10.1016/0005-2787(72)90159-1

Cited by 53 publications

(15 citation statements)

References 38 publications

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“…Third, the protein composition of P2 falls in the range expected for wheat germ ribosomes (Bielka, 1982). Fourth, the weight ratio between RNA and protein content in P2 is also consistent with the reported data of ribosomes (Cammarano et al, 1972). Fifth, both the activity in P2 and the activity in ribosomes show similar sensitivity to thermal and alkaline treatment.…”

Section: Discussionsupporting

confidence: 86%

“…This value is consistent with a molecular mass of 3.9 × 10 6 daltons for plant ribosomes (Cammarano et al, 1972) and of 3.8 × 10 6 daltons for Artemia salina (brine shrimp) ribosomes (Nieuwenhuysen & Clauwaert, 1978). Second, the RNA composition of P2 is also consistent with wheat germ ribosomal RNAs (Cammarano et al, 1972). Third, the protein composition of P2 falls in the range expected for wheat germ ribosomes (Bielka, 1982).…”

Section: Discussionsupporting

confidence: 79%

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Role of Ribosomes in Reinitiation of Membrane Insertion of Internal Transmembrane Segments in a Polytopic Membrane Protein

et al. 1997

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The topogenesis of membrane proteins with a single transmembrane (TM) segment is well understood. However, understanding the topogenesis and membrane assembly of membrane proteins with multiple TM segments (polytopic) is still incomplete. Recently, several studies on P-glycoprotein (Pgp) suggested that the topogenesis of polytopic membrane proteins is likely more complicated than anticipated. While studying the mechanism by which Pgp topogenesis is determined, we unexpectedly found that ribosomes or proteins associated with ribosomes are involved in regulating the membrane insertion and folding of Pgp during its translation. We discovered that when Pgp was translated by wheat germ ribosomes in vitro, TM3 could not reinitiate the insertion of the protein into microsomal membranes following the membrane insertion of TM1 and TM2. In contrast, TM3 could reinitiate membrane insertion when the protein was translated by rabbit reticulocyte ribosomes. These findings suggest that ribosomes or proteins associated with ribosomes play an important role in membrane insertion and folding of TM segments of Pgp and that rabbit reticulocyte and wheat germ ribosomes may use different mechanisms to control the membrane insertion of the same nascent peptide. We propose that ribosomes or proteins associated with ribosomes help reinitiate insertion of internal TM segments into the membrane by dissociation and reassociation with the protein-conducting channel in ER membranes.

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Section: Discussionsupporting

confidence: 86%

Section: Discussionsupporting

confidence: 79%

Role of Ribosomes in Reinitiation of Membrane Insertion of Internal Transmembrane Segments in a Polytopic Membrane Protein

et al. 1997

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“…Many studies on eukaryotic ribosomes and their derivatives have relied on the assumption that RNA/protein ratios can be calculated from equilibrium buoyant densities of the particles in CsCl (4)(5)(6)(7). I demonstrate here that this assumption, as generally used, is wrong.…”

mentioning

confidence: 83%

“…Subunits were fixed in 6% formaldehyde, 50 mM triethanolamine -HCl, pH 7.6, 100 mM KCl, 1 mM MgCl2 for 2-3 hr, then dialyzed overnight against 1% formaldehyde, 25 mM triethanolamine-HCl, pH 7.6, 50 mM KCl (7). CsCl (Harshaw optical grade) was dissolved in the latter buffer, and preformed linear gradients were prepared with the sample contained in the lighter CsCl solution.…”

Section: Methodsmentioning

confidence: 99%

Composition of Mammalian Ribosomal Subunits: A Re-Evaluation

McConkey

1974

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

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A method for preparation of highly active mammalian ribosomal subunits is described, which yields 60S subunits containing no more than 33% protein. It is suggested that the composition of these subunits corresponds closely to that of Escherichia coli 50S subunits. Data on the composition of bacterial and mammalian ribosomal subunits recovered from CsCl are given. It is shown that the commonly employed assumptions about the relationship between the buoyant density of a particle in CsCl and its protein content are in error. The composition of ribosomal subunits cannot ordinarily be calculated from the buoyant density in CsCI.The fundamental aspects of protein synthesis in eukaryotes appear to be virtually the same as in prokaryotes; but, surprisingly, eukaryotic ribosomes have often been reported to contain significantly more proteins than bacterial ribosomes (1-3). In this report I shall show that it is possible to prepare active mammalian ribosomal subunits in such a way that the large (60S) subunits contain the same number of proteins as the homologous 50S subunits of Escherichia coli. This leads to the suggestion that there is no fundamental difference in the composition of large ribosomal subunits of bacteria and mammals, although there is a marked difference in size.Many studies on eukaryotic ribosomes and their derivatives have relied on the assumption that RNA/protein ratios can be calculated from equilibrium buoyant densities of the particles in CsCl (4-7). I demonstrate here that this assumption, as generally used, is wrong. Mammalian and bacterial ribosomal subunits retain less protein in CsCl than has been assumed by others, and subunits with widely different buoyant densities may have the same overall composition. MATERIALS AND METHODSPreparation of Ribosomal Subunits. Livers were obtained from male black C57 mice that had been starved overnight. HeLa cells were grown in Eagle's Minimum Essential Medium (8) in spinner culture containing 5% calf serum. The standard "high-salt" isolation procedure for ribosomal subunits was based on the method of Blobel and Sabatini (9). Livers or HeLa cells were homogenized in 10 mM Tris * HCl, pH 7.4, 10 mM KCl, 1 mM MgCI2, 1 mM dithiothreitol, using 10 ml of buffer per gram of tissue. One-tenth volume of 3 M KCl, 20 mM MgCI2 was added slowly to disrupt cytoplasmic aggregates and improve yield of ribosomes. Nuclei and mitochondria were removed by centrifugation at 15,000 X g for 15 min. The supernatant was then adjusted to 0.5% Brij 58 and 0.5% sodium deoxycholate. Ribosomes were sedimented at 40-50,000 rpm overnight in the Tvpe 65 Beckman rotor through a cushion consisting of 1.75 M sucrose, 50 mM TrisHCl, pH 7.4, 100 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol. Ribosome pellets were suspended in cushion buffer lacking sucrose and treated with 5 X 10-i M puromycin for 15 min at 37°. Subunits were then separated on 10-30% sucrose gradients containing 500 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl, pH 7.4, and 1 mM dithiothreitol. Subunits were harvested by centrifugation,...

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“…ucidophilu ribosomal subunits were obtained according to Cammaranoet al [18]; the L-rRNAand S-rRNA species from all sources were isolated as described previously [8]. RNA concentrations were estimated assuming a specific absorption.…”

Section: Gronvh Of Bucteriu and L~ihelling Conditionsmentioning

confidence: 99%

Characterization of the Secondary Structure Features of Escherichia coli, Caldariella acidophila and Mammalian Ribosomal RNA Species by Chemical Modification of Sterically Exposed Bases

Cammarano

Londei

Biagini³

et al. 1982

European Journal of Biochemistry

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The helix content of rRNA species (Escherichiu coli, Calduriellu ucidophila, rat liver) and the G . C content of their bihelical domains have been investigated by chemical modification of uracil and cytosine residues with probes specific for sterically exposed bases. By using radioactively labelled rRNA, G . C base pairs and the sum of A . U plus G + U base pairs have been quantified assuming that they are numerically identical with the unreactive cytosine and uracil rings, respectively. Exposed uracil bases were probed by their conversion to alkali-labile. nonultraviolet-absorbing sulphonated adducts, with 1.33 M bisulfite pH 7, at 20 "C; the adducts can be separated from unreacted uracil, and quantified, by cation-exchange chromatography of RNase T2 plus pancreatic RNase digests of bisulfite-modified rRNA. Exposed cytosines were probed by their conversion to methoxyaminated, alkalistable, derivatives with 1 M methoxyamine. pH 5.5. at 37'C, and quantified by monitoring the CMP/AMP radioactivity ratio after alkaline hydrolysis of modified rRNA. Exposed uracil rings can also be estimated spectrophotometrically by the alkali-catalyzed reversal of the non-ultraviolet-absorbing sulphonated adducts after separation of the latter from unreacted uracil. The cytosine deamination reaction, catalyzed by bisulfite at pH 6, has also been investigated and found to exhibit little specificity for sterically exposed bases of rRNA, the (G + C)-richer rRNA species of C. ucidophila being considerably less susceptible to non-specific deamination than the (G + C)-poorer rRNA of E. coli. A high degree of congruence is shown to exist between results obtained by chemical modification and melting hyperchromicity experiments.The principal secondary structure features of RNA species can be evaluated spectrophotometrically by melting hyperchromicity methods 11-31. The amount of bihelical secondary structure (i.e. the fraction of residues involved in hydrogen-bonded base-pairing) can be estimated by comparing the hypochromism of the partly double-helical polynucleotide to that of an appropriate fully bihelical analogue to denaturation from unpaired but stacked bases; solvents used to discriminate single-stranded stacking from helix to coil transitions, such as guanidinium chloride [7-91, may Ahhreviarrons. S-rRNA and L-rRNA indicate the RNA species isolated from the smaller and the larger ribosomal subunit, respectively: U* and U . SO3 are used, as convenient. to designate 5.6-dihydrouracil 6-sulphonate: C*, rnethoxyaminated cytosine.Enzymes. Pancreatic RNase (EC 3.1.27.5); T2 KNase (EC 3.1.27.1).also cause denaturation of bihelical segments to an extent depending on both solvent concentration and bihelix stability (Cammarano et al., unpublished work); this may constitute a source of error in investigating eukaryotic mRNA species, whose long poly(A) tails are fully stacked in neutral salt solutions; (d) unless specially built devices are used [lo], the potential of melting hyperchromicity methods is often limited by the availability of m...

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Size heterogeneity of the large ribosomal subunits and conservation of the small subunits in eucaryote evolution

Cited by 53 publications

References 38 publications

Role of Ribosomes in Reinitiation of Membrane Insertion of Internal Transmembrane Segments in a Polytopic Membrane Protein

Role of Ribosomes in Reinitiation of Membrane Insertion of Internal Transmembrane Segments in a Polytopic Membrane Protein

Composition of Mammalian Ribosomal Subunits: A Re-Evaluation

Characterization of the Secondary Structure Features of Escherichia coli, Caldariella acidophila and Mammalian Ribosomal RNA Species by Chemical Modification of Sterically Exposed Bases

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