Photoincorporation of puromycin into rat liver ribosomes and subunits

Abstract

[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.

Affinity labeling of the P site of Drosophila ribosomes: a comparison of results from (bromoacetyl)phenylalanyl-tRNA and mercurated fragment affinity reactions

“…ribosomal proteins run on 2-D gels (Reboud et al, 1981;Grant et al, 1979). Similarly, we noted that approximately 10% of the loaded radioactivity was recovered on the 2-D acid/SDS gels and 3-7-month exposure times were necessary for autoradiography.…”

Partial reassembly of active 60S ribosomal subunits from rat liver following treatment with dimethylmaleic anhydride

“…Addition of the split protein fraction to the residual cores restored to a large extent all the catalytic activities quoted above. Our reconstituted subunits had almost the same biophysical properties as the control 60s subunits : density, protein pattern, sedimentation profiles, ability to associate with 40s subunits (see Figs 1 -3 ) and also melting-out [5] external [I81 cross-linked to LA33 [16] external [18] cross-linked to LA33 [I61 external [5] cross-linked to L24 and L38 [I91 close to A and P sites [5,16,23,24,251 + [281 + cross-linked to S3a [16] cross-linked to EF-2 [16] + cross-linked to S6 [I61 external [18] external [5] cross-linked to P1 [19] + [27] exchangeable with cross-linked supernatant to EF-2 (P2, [16]) protein [29] curves (Lavergne, J. P., unpublished results). All our results taken together proved that the 15000 mol/mol DMMA split proteins play a part in the conformation of the large subunit and in the interaction of this with EF-2, the 40s subunits and possibly the aminoacyl-tRNA.…”

Identification by RNA-protein cross-linking of ribosomal proteins located at the interface between the small and the large subunits of mammalian ribosomes.

“…Initiation factor eIF-3 has been found to attach to the 40S subunit close to the interface region (Emanuilov et al, 1978) in contact with proteins S3 arid S4 among others (Nygard and Westermann, 1982). Proteins S3, S3a, and S6 have been found in close contact with both artificial and physiological messengers (Terao and Ogata, 1979;Stahl and Kobets, 1981;Reboud et al, 1981;Takahishi and Ogata, 1981). These proteins have been further designated as presumptive P-site proteins because of their ability to cross-link to initiator tRNA .…”

“…Owing to this functional cooperativity, a number of active ribosomal domains would be expected to occur in the contact region between the subparticles. In mammalian ribosomes, mRNA (Nonomura et al, 1971;Stahl and Kobets, 1981;Reboud et al, 1981;Takahishi and Ogata, 1981), initiation factor eIF-3 (Emanuilov et al, 1978, Nygard andWestermann, 1982), and the A and P sites of the peptidyltransferase center (Metspalu et al, 1978;Ulbrich et al, 1980a;Todokoro et al, 1981) are supposed to be associated with the contact region. By analogy with the situation in bacteria (Girshovich and Kurtskhalia, 1978), the attachment site for translocation factor EF-2 is probably also located in this region.…”