Resistance of messenger RNA-bound ribosomes to proteolytic dissociation

Freedman, Michael L.; Vélez, Ramón; Mucha, Jitka

doi:10.1016/0014-4827(72)90011-0

Cited by 9 publications

(6 citation statements)

References 19 publications

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“…410 dissociation into subunits, confirming results obtained in previous studies (Malkin & Rich, 1967;Velez et al, 1971;Freedman et al, 1972).…”

Section: Resultssupporting

confidence: 88%

“…Mild low-temperature proteolysis with Pronase selectively dissociates ribosomes not attached to mRNA into 40S and 60S subunits (Velez etal., 1971). Ribosomes attached to either intact or fragmented mRNA are completely resistant and remain intact (Velez et al, 1971;Freedman et al, 1972). Monoribosomes attached to mRNA resist proteolytic dissociation as well as polyribosomes do (Freedman et al, 1972).…”

Section: Discussionmentioning

confidence: 99%

“…Ribosomes attached to either intact or fragmented mRNA are completely resistant and remain intact (Velez et al, 1971;Freedman et al, 1972). Monoribosomes attached to mRNA resist proteolytic dissociation as well as polyribosomes do (Freedman et al, 1972). This protective effect of mRNA attachment is independent of the presence of nascent chains on the ribosomes (Freedman et al, 1972).…”

Section: Discussionmentioning

confidence: 99%

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Monoribosomal attachment to messenger ribonucleic acid in sodium fluoride-treated rabbit reticulocytes

Geraghty

Galler

Schiffman

et al. 1973

Biochemical Journal

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“…410 dissociation into subunits, confirming results obtained in previous studies (Malkin & Rich, 1967;Velez et al, 1971;Freedman et al, 1972).…”

Section: Resultssupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Monoribosomal attachment to messenger ribonucleic acid in sodium fluoride-treated rabbit reticulocytes

Geraghty

Galler

Schiffman

et al. 1973

Biochemical Journal

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“…When we investigated the effect of ferritin on the ribosome-polyribosome component of the lysates, it was observed that there was a disaggregation of polyribosomes to monoribosomes and ribosomal subunits. While this could be interpreted as evidence for a block in initiation (Rabinovitz et al, 1969) at the step of attachment of the 40s subunit to mRNA (Freedman & Rabinovitz, 1970), it could also be that the ferritin was contaminated with a proteolytic enzyme which causes dissociation of ribosomes into subunits (Velez et al, 1971 ;Freedman et al, 1972). To investigate this possibility, the preparations of ferritin were assayed for protease activity.…”

Section: Discussionmentioning

confidence: 99%

Ferritin and Sideroblastic Anaemias: Inhibition of Protein Synthesis by Protease Contaminants in Commercial Preparations of Ferritin

Freedman¹,

Cohen²,

Rosman³

et al. 1976

Br J Haematol

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Commercial preparations of ferritin inhibited reticulocyte-lysate cell-free protein synthesis and disaggregated polyribosomes to monoribosomes and ribosomal subunits. These effects were prevented by addition of reduced glutathione (GSH) to the incubation medium, but ferritin did not lower GSH concentration in the lysates. The more purified the ferritin preparation, the less inhibition of protein synthesis was observed. These data suggested that the effect was due to a contamination of the ferritin with proteolytic activity. In confirmation of this proposal we demonstrated that there was protease activity in both the 2X and 5X crystalized ferritin preparations, with 2.5 times greater activity in the 2X preparation. The proteolytic activity in ferritin was inhibited by incubation with the protease inhibitor tosyl lysine chloromethyl ketone (TLCK). When an amount of trypsin equivalent to the protease activity of the ferritin was added to the incubation mixture, similar effects on protein synthesis and the ribosome-polyribosome component were found. Both GSH and TLCK prevented these effects of trypsin. These data suggest that the previously reported effect of ferritin on reticulocyte cell-free protein synthesis was due to contamination of the ferritin by a protease. It appears that ferritin does not play a direct role in the pathogenesis of sideroblastic anaemias.

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“…Third, one could separate monosomes into free and mRNAbound ribosomes by density centrifugation ( 11) or selective proteolytic dissociation (12). We hope to carry out some of these investigations in conjunction with tests involving inhibitors and radioactive precursors in an attempt to find out if the increase in polysome-associated mRNA is due to its synthesis, and whether these increases in available mRNA templates are accompanied by changes in ribosome initiation.…”

Section: And Discussionmentioning

confidence: 99%

Polyribosomes from Peas

Davies¹

1973

Plant Physiol.

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Polyribosomes as large as 10-mers (strands of messenger RNA bearing 10 ribosomes) were isolated from etiolated pea (Pisum sativum L. var. Alaska) stem tissue during all stages of development when methods were used which essentially eliminated ribonuclease activity during extraction. Actively growing tissue, harvested from the apical 10 mm, yielded many large polyribosomes and a low (<20%) proportion of monosomes. Similar tissue, allowed to age by applying lanolin to decapitated apices, showed a progressive decrease in number of larger polyribosomes and an increase in the proportion of monosomes. Hormone treatments, which prolonged growth and delayed aging, delayed the loss in large polyribosomes and the increase in proportion of monosomes. Growth-stimulating hormones, added to previously aged tissue, stimulated the production of many large polyribosomes in pre-existing ceils.It is suggested that (a) large polyribosomes occur in all regions of the pea stem, (b) changes in polyribosome distribution appear to precede changes in growth rate, (c) loss of larger polyribosomes is closely related to a decrease in mRNA templates followed more gradually by loss of ribosomes, (d) hormone-stimulated continuation of growth is accomplished through maintenance of available mRNA.Methods are described, involving detailed analysis of polysome distribution, which, although they cannot be used to measure changes in initiation of ribosomes on to mRNA, do permit measurement of the amount of polysomal-associated mRNA present in tissues at different stages of growth. These analyses lead to the further suggestion that hormone stimulation of growth of previously nongrowing tissue is accomplished primarily through an increase in available mRNA prior to synthesis of ribosomes.In an earlier paper (7), we showed that high yields of large polyribosomes could be obtained from actively growing, etiolated pea stem tissue, homogenized in media buffered with tris-HCl at high concentration (200 mM) and pH (8.5), and that the effectiveness of the buffer was due to its prevention of RNase activity during extraction. We MATERIALS AND METHODSSeedlings of Pisum sativum L. var. Alaska were grown in darkness at 23 to 25 C for 7 to 8 days until the third internode was 2 to 4 cm long (7). In some instances, tissue was excised from different regions of untreated plants. In other instances, plants were decapitated, a mark was made 10 mm below the apex to delineate a segment of tissue, and lanolin with or without additives was applied to the cut end. The segments were excised after various time periods. All operations were conducted under dim green light. Polyribosomes were isolated as described earlier (7) by homogenizing tissue in a mortar in 5 to 10 volumes of grinding buffer (0.25 M sucrose; 0.2 M tris-HCl, pH 8.5; 60 mm KCl, and 30 mm MgCl2). The resulting brei was centrifuged at 30,000g for 20 min. The supernatant was layered on a 4-ml pad of 1.5 M sucrose in gradient buffer (40 mm tris-HCl, pH 8.5; 20 mM KCl; 10 mm MgCl2) and centrifuged at 1...

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Resistance of messenger RNA-bound ribosomes to proteolytic dissociation

Cited by 9 publications

References 19 publications

Monoribosomal attachment to messenger ribonucleic acid in sodium fluoride-treated rabbit reticulocytes

Monoribosomal attachment to messenger ribonucleic acid in sodium fluoride-treated rabbit reticulocytes

Ferritin and Sideroblastic Anaemias: Inhibition of Protein Synthesis by Protease Contaminants in Commercial Preparations of Ferritin

Polyribosomes from Peas

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