The synthesis of a photoreactive puromycin analogue and its application for labeling proteins in the 50-S subunit of Escherichia coli ribosomes.

A photoreactive puromycin analogue, 6-dimethylamino-9-[3-(p-azido-L-beta-phenylalanylamino)-3-deoxy-beta-ribofuranosyl] purine, was synthesized. Biological activity was demonstrated by inhibition of the poly (U)-directed phenylalanine-incorporation system and by decomposition of isolated polysomes from Escherichia coli. The 3H-labeled puromycin analogue was covalently attached to the 50-S subunit of isolated 70-S ribosomes from Escherichia coli after irradiation. More than 90% of the radioactivity was bound to the protein fraction. The 70-S proteins were separated by two-dimensional gel electrophoresis. The proteins labeled primarily were those of the 50-S subunit, identified as L6, L13, L18, L22 and L25. On the basis of the affinity label used and supportive data from the literature, it is concluded that these proteins are at the active center of the 50-S particle and probably belong to the region of the ribosomal A site.