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 rRNA. The results from simultaneous 1-dimensional and 2-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. 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. Proteins S3 and perhaps S23-24, which were significantly labeled in crude ribosomes too, may also belong to a specific site interacting with puromycin is discussed.