[3H]Puromycin was covalently incorporated into rat liver ribosomes and isolated 40S and 60S subunits on irradiation at 254 nm. A study of the concentration dependence of this photolytic incorporation suggested that it arose from specific sites on isolated subunits but also from unspecific ones in the case of ribosomes, these sites being probably located on contaminant nonribosomal proteins. Puromycin was incorporated simultaneously into ribosomal proteins and rRNAs. The results from simultaneous one-dimensional and two-dimensional gel electrophoreses showed a small distribution of label among ribosomal proteins in 60S subunits and in 80S ribosomes, L10 being the most radioactive protein. Some antibiotics, which act on the peptidyltransferase center (amicetin and gougerotin), and also tetracycline competed with this labeling. Therefore, it was concluded that puromycin interaction with protein L10 occurred most likely at a functional site. In the case of free 40S subunits, labeling distribution among proteins was much wider. The possibility that proteins S3 and perhaps S23-24, which were significantly labeled in crude ribosomes too, also belong to a specific site interacting with puromycin is discussed.
Irradiation at 254 nm of the rat liver 40 S ribosomal subunit—poly(U)—Phe[32P]tRNA complex induced a covalent linkage of tRNA with a limited number of ribosomal proteins. After RNA hydrolysis, S10 was found to be the protein most highly labeled by radioactive nucleotides. Some radioactivity was also associated with protein S6‐6a, S3a, S2 and S13–15.
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...
[3H]Tetracycline was covalently incorporated into rat liver ribosomes and isolated 40‐S and 60‐S subunits on irradiation at 254 nm. The antibiotic was almost exclusively incorporated into ribosomal proteins. At least some of these proteins are assumed to be involved in ribosomal function, since photoincorporated tetracycline was found to inhibit the activity of 40‐S and 60‐S subunits in the poly(U)‐directed protein‐synthesizing system as well as that of the 40‐S subunit in the poly(U)‐mediated [14C]Phe‐tRNA binding. The results from simultaneous one‐dimensional and two‐dimensional gel electrophoreses showed a small distribution of label among ribosomal proteins in 60‐S subunits and in 80‐S ribosomes, L10 being the most radioactive protein. As non‐acylated tRNA partly competed with this labeling, it is likely that tetracycline interaction with these proteins occurred at a functional site. L10 has already been found to interact with puromycin [Reboud, A. M., Dubost, S., Buisson, M. & Reboud, J. P. (1981) Biochemistry, 20, 5281–5288]. In the case of free 40‐S subunits the label distribution was wider among ribosomal proteins. No particular role has yet been found for the most labeled protein, S12, but protein S3a, which was also highly labeled, has already been reported to be involved in subunit function.
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