The Dissociation of Rabbit Reticulocyte Ribosomes with EDTA and the Location of Messenger Ribonucleic Acid

Abstract

Rabbit reticulocyte ribosomes were incubated with polyuridylic acid a t 37" for 1 h under conditions suitable for protein synthesis. Ribosomes containing bound polyuridylic acid were isolated and dissociated into subparticles by treatment with EDTA. The subparticles were separated by zone centrifugat,ion in a sucrose density gradient and the distribution of polyuridylic acid was examined by measuring the ability of gradient fractions to stimulate the incorporation of [14C]phenylalanine into protein by a cell-free system. It was observed that the peak of polyuridylic acid activity corresponded to a region of the subparticle gradient having a sedimentation coefficient of about 50 S. When [14C]polyuridylic acid was used in the initial binding reaction, subsequent analysis of subparticles produced by treatment with EDTA confirmed the presence of polyuridylic acid on or close t o the large ribosomal subparticle.Bacterial ribosomes can be dissociated readily into two subparticles and messenger RNA by reducing the magnesium concentration of the medium [1,2], but mammalian ribosomes appear to be more stable. The conditions under which they can be dissociated have been investigated by a number of workers who have shown that treatment with EDTA [3] or pyrophosphate [4], or exposure to high pH [5] is required.The fate of mRNA when mammalian ribosomes are dissociated into subparticles is uncertain. On the one hand, Huez et al. [6] reported that treatment of reticulocyte polysomes with EDTA releases a minor RNA component which is rapidly labelled in vivo and sediments with a coefficient of 9-10 S. On the basis of sue, kinetics of labelling and other properties this RNA is considered to be haemoglobin messenger [7], but it should be noted that it has not yet been shown to have any stimulatory activity for cell-free protein synthesis. On the other hand, stimulatory RNA was recovered in the ribosomal subparticles after dissociation of reticulocyte ribosomes, most of the stimulatory activity being associated with the RNA from the large (60 S) subparticle [5]. The validity of using stimulation of cell-free protein synthesis as a criterion of messcnger activity has recently been questioned by Hunt and Wilkinson[8] who showed that the synthesis of a haemoglobin-like product by a cell-free preparation from reticulocytes was stimulated by liver as well as by reticulocyte RKA. It is possible, therefore, that cell-free protein I n view of these uncertainties, we decided to investigate the fate of a synthetic messenger, poly U, after interaction with reticulocyte ribosomes in a cell-free protein-synthesizing system and subsequent dissociation of the ribosome-poly U complex into subparticles by treatment with EDTA. This approach has the advantage that the messenger can be identified readily, either by its ability to synthesize a characteristic polypeptide or by interacting ribosomes with radioactive poly U. Both methods were used in the present experiments, which lead to the conclusion that a substantial part of the poly U is not rel...

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

“…Because we routinely spin samples at 16,000 x g before SEC and given the ultracentrifugation profile of nonvesicular exRNAs ( Figure 3, H-I ), the Ca 2+ /Mg 2+ -induced loss of the P0 peak can be explained by a disassembly of higher order ribosomal aggregates in the absence of divalent cations. This is consistent with the involvement of magnesium ions in the stabilization of ribosomes and polysomes and their disassembly upon EDTA treatment 48 , although SEC-purification of polysomes is possible under optimized conditions 49 .…”

Structure/Function Analysis of Tristetraprolin (TTP): p38 Stress-Activated Protein Kinase and Lipopolysaccharide Stimulation Do Not Alter TTP Function

“…8). The presence of EDTA, which disrupts polyribosomes (34), results in a loss of TTP and eIF4E localization to these dense fractions, confirming the polysomal localization of TTP (Fig. 8).…”

Structural transformations of ribosomes (dissociation, unfolding and disassembly)

“…Among them, the increase of the ionic strength at the expense of monovalent cations (K', NH:, etc.) is well known to promote dissociation [2,4,6,9,24,25,[38][39][40][41][42][43] ] . Na' and Li', even in relatively low concentrations, have an especially strong dissociating effect [6, .…”