[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
| Autor: | Clifford C. Hall, Barry S. Cooperman, Deanna Johnson |
|---|---|
| Rok vydání: | 1988 |
| Předmět: |
Photoaffinity labeling
Eukaryotic Large Ribosomal Subunit Immunoelectron microscopy Chromosome Mapping Nucleic Acid Hybridization Affinity Labels Ribosomal RNA Biology Biochemistry Molecular biology Ribosome RNA Ribosomal 23S RNA Ribosomal 23S ribosomal RNA Escherichia coli Autoradiography Nucleic Acid Conformation Electrophoresis Polyacrylamide Gel Puromycin Eukaryotic Ribosome 50S |
| Zdroj: | Biochemistry. 27:3983-3990 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00411a014 |
| Popis: | 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) |
| Databáze: | OpenAIRE |
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