Photoaffinity labeling of Escherichia coli ribosomes by an aryl azide analogue of puromycin. Evidence for the functional site specificity of labeling.
The photoincorporation of p-azido[3H]puromycin [6-(dimethylamino)-9-[3'-deoxy-3'-[(p-azido-L-phenylalanyl)amino]-beta-D-ribofuranosyl]purine] into specific ribosomal proteins and ribosomal RNA [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry (preceding paper in this issue)] is decreased in the presence of puromycin, thus demonstrating that labeling is site specific. The magnitudes of the decreases in incorporation into the major labeled 50S proteins found on addition of different potential ribosome ligands parallel the abilities of these same ligands to inhibit peptidyltransferase. This result provides evidence that p-azidopuromycin photoincorporation into these proteins occurs at the peptidyltransferase center of the 50S subunit, a conclusion supported by other studies of ribosome structure and function. A striking new finding of this work is that puromycin aminonucleoside is a competitive inhibitor of puromycin in peptidyltransferase. The photoincorporation of p-azidopuromycin is accompanied by loss of ribosomal function, but photoincorporated p-azidopuromycin is not a competent peptidyl acceptor. The significance of these results is discussed. Photolabeling of 30S proteins by p-azidopuromycin apparently takes place from sites of lower puromycin affinity than that of the 50S site. The possible relationship of the major proteins labeled, S18, S7, and S14, to tRNA binding is considered.
D000345:Affinity Labels / Q000378:metabolism; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008433:Mathematics; D010777:Photochemistry; D011691:Puromycin / Q000031:analogs & derivatives* / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000032:analysis*
D000345:Affinity Labels; D012269:Ribosomal Proteins; D011691:Puromycin; D012313:RNA; C017044:4-azidopuromycin