Localization of the site of 23S RNA photoaffinity labeling by [3H]-p-azidopuromycin. Additional evidence that domain V contains the peptidyl transferase center

The photoincorporation of [3H]-p-azidopuromycin, a photolabile functional analogue of puromycin, into the RNA portion of 70S ribosomes was analyzed by hybridization of photoaffinity-labeled rRNA to restriction fragments of complementary DNA derived from plasmid pKK3535. The method of analysis was as previously described, except that a double-labeling approach, using uniformly labeled [14C]-rRNA, was employed to correct for differences in the yields of specific hybrids. Approximately 3/4 of the labeling was found in 23S rRNA, with the major site of labeling falling within bases 2445-2668, in region V of 23S rRNA. Such labeling was decreased in the presence of puromycin, providing evidence that it occurs from the peptidyl transferase center. Efforts to localize the site of p-zaidopuromycin labeling to a specific base within this region, using an approach based on the interference of a photoaffinity labeled base with reverse transcriptase activity, are underway