[3H]-p-azidopuromycin photoaffinity labeling of Escherichia coli ribosomes: evidence for site-specific interaction at U-2504 and G-2502 in domain V of 23S ribosomal RNA.
Previously we (1) showed that [3H]-p-azidopuromycin was a functional puromycin analogue that, on photolysis in the presence of 70S ribosomes from Escherichia coli, photoincorporated site specifically into proteins L23, L18/22, and L15 [Nicholson, A.W., Hall, C.C., Strycharz, W.A., & Cooperman, B.S. (1982) Biochemistry 21, 3809-3817] and (2) used immunoelectron microscopy to localize the principal sites of p-azidopuromycin photoincorporation within the 50S subunit [Olson, H.M., Nicholson, A.W., Cooperman, B.S., & Glitz, D.G. (1985) J. Biol. Chem. 260, 10326-10331]. These studies are here continued by identification of the principal sites of [3H]-p-azidopuromycin photoincorporation into ribosomal RNA. The major portion of such photoincorporation, 72%, takes place into 23S rRNA. Analysis by hybridization of the photoaffinity-labeled rRNA to restriction enzyme fragments of plasmid pKK3535, which contains rrnB DNA, using a refinement of a recently developed methodology [Hall, C.C., Smith, J.E., & Cooperman, B.S. (1985) Biochemistry 24, 5702-5711], shows that the most prominent [3H]-p-azidopuromycin photoincorporation occurs within bases 2445-2668 in domain V [Noller, H.F. (1984) Annu. Rev. Biochem. 53, 119-162] of 23S rRNA. Photoincorporation into this region is site specific, as demonstrated by the decrease in photoincorporation of radioactivity when unlabeled puromycin is included in the photolysis solution. Significant site-specific photoincorporation also occurs within bases 489-681 in domain II of 23S RNA. Further localization, by the method of reverse transcriptase primer extension [Barta, A., Steiner, G., Brosius, J., Noller, H.F., & Kuechler, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3607-3611], provides evidence that U-2504 and G-2502 are the principal sites of p-azidopuromycin interaction.(ABSTRACT TRUNCATED AT 250 WORDS)
D000345:Affinity Labels / Q000494:pharmacology*; D001345:Autoradiography; D002874:Chromosome Mapping; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D011691:Puromycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D012335:RNA, Ribosomal / Q000235:genetics*; D012338:RNA, Ribosomal, 23S / Q000235:genetics*
D000345:Affinity Labels; D012335:RNA, Ribosomal; D012338:RNA, Ribosomal, 23S; D011691:Puromycin; C017044:4-azidopuromycin