Characterization and properties of a 5S‐RNA‐protein complex released from heated 60S ribosomal subunits

Abstract

When rat liver 60s ribosomal subunits were heated in phosphate buffer in the presence of MgCI,, 5s RNA was released in the form of a nucleoprotein complex (RNP,), which was isolated either by electrophoresis in polyacrylamide gel or centrifugation through a sucrose gradient. In addition to L5 several proteins of functional significance were identified in the complex : the acidic phosphoproteins P1-P2 and, as weaker spots, L3-L4, L6-L7 and L22. Most of these proteins were also found, but only as traces, in the RNP,?,,, used as a control. RNP, was able to associate with 40s subunits. Our results support the interpretation that RNPH is located at the subunits' interface, at or near the peptidyl-transferase center.5s RNA, a component of the large 50s and 60s ribosomal subunits, is required for protein-synthetic activity; but its precise role is still unknown. It has been postulated that it has specific functional roles in the ribosomal peptidyltransferase or GTPase/ATPase activities, a tRNA-binding function and a role in subunit association. This last hypothesis was mainly based on an observed sequence complementation between 5s rRNA and the 3' termini of 18s rRNA (for references see [l]).The 5s RNA is integrated into the large ribosomal subunit in a complex fashion, associated with specific proteins. These have been identified by binding assays on nitrocellulose filter, affinity chromatography or analysis of the 5s-RNA -protein complex (RNP) released from subunits exposed to chemical compounds such as EDTA, the most widely used agent, high salt concentrations, citrate, formamide or urea. While all published studies agree and indicate the existence of multiple well-defined SS-RNA-binding proteins in prokaryotes, there is still some controversy about the number and nature of the proteins interacting with eukaryotic 5s RNA. Thus, in the case of rat liver ribosomes, a single protein (L5) or at most two proteins (L5, L7 see Discussion) have been identified in the RNPEDTA [2 -61 while additive proteins with affinities for 5s RNA have been found by other methods (proteins L6, L19,This report describes the purification of a SS-RNA -protein complex (RNPH) released from heated rat liver 60s ribosomal subunits. Proteins present in RNPH were identified, and the complex was shown to interact with 40s ribosomal subunits. MATERIALS AND METHODS Preparation of ribosomes and ribosomal subunitsRibosomes were prepared as described by Moldave [l 11. Ribosomal subunits were prepared from free rat liver polysomes, freed of ferritin by precipitation with magnesium, according to the KCl/puromycin procedure adapted from Blobel and Sabatini [12] as previously reported [13]. 5s-RNA -protein complex preparationAliquots of 60s ribosomal subunits (6 A260 units/O.l ml buffer A consisting of 1 mM potassium phosphate, pH 7.4, 30 mM KC1, 5 mM MgCl, and 20 mM 2-mercaptoethanol) were heated as described in Results. The small-molecular-mass material (RNP,) released under these conditions was isolatzd either by one-dimensional gel electrophoresis using...

Photo‐induced protein cross‐linking to 5S RNA and 28–5.8S RNA within rat‐liver 60S ribosomal subunits

“…As in these reports, we did not find protein L 5 which, according to Terao et al [27], is the only one to be cross-linked to 5s RNA within the RNPEDTA released from ultravioletirradiated 6 0 s subunits. This is probably because L5 is very poorly soluble [8]. The discrepancies between our crosslinking results and the results reported above are certainly due to the fact that we identified the proteins of the whole 5 s RNA fraction whereas there is no certainty that EDTA can release all 5 S-RNA -protein complex from irradiated subunits as it does from control subunits.…”

Μελετες Επι Της Πεπτιδυλοτρανσφερασης Σε Συστημα Απο Δικτυοερυθροκυτταρα Κουνελιου

“…7. Το 5S rRNA στο μοντέλο αυτό έχει τοποθετηθεί κοντά στην πρωτεΐνη L5, διότι έχει αποδειχθεί, ότι η L5 αποτελεί μέρος του ριβονουκλεοπρωτεινικού συμπλόκου 5S RNP (σύμπλοκο του 5S rRNA με ριβοσωματικές πρωτεΐνες), (Terao et al 1975, Reboud et al 1984, Takahashi and Ogata 1985, το οποίο ελευθερώνεται με διάφορες μεθόδους από την 60S υπομονάδα. Επίσης η L5 μπορεί και αντιδρά με το 5S rRNA της μεγάλης υπομονάδας σε πειράματα επιχιασμού (Terao et al 1980).…”