Reconstitution of Escherichia coli ribosomes containing puromycin-modified S14: functional effects of the photoaffinity labeling of a protein essential for tRNA binding.
In previous work we have shown that puromycin photoaffinity labels two proteins, L23 and S14, from separate sites of high affinity on Escherichia coli ribosomes [Jaynes, E. N., Jr., Grant, P. G., Giangrande, G., Wieder, R., & Cooperman, B. S. (1978) Biochemistry 17, 561-569; Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274], that puromycin-modified S14 is separable from native S14 by reverse-phase high-performance liquid chromatography (RP-HPLC), and that ribosomal proteins prepared by RP-HPLC can be reconstituted into active 30S subunits [Kerlavage, A. R., Weitzmann, C. J., & Cooperman, B. S. (1984) J. Chromatogr. 317, 201-212]. In this work we definitively identify puromycin-modified S14 by tryptic fingerprinting, an analysis that also provides evidence that the single tryptophan-containing peptide in S14 is the site of puromycin photoincorporation. We show that reconstituted 30S subunits, in which all of the S14 present is stoichiometrically modified with puromycin and all other ribosomal components are present in unmodified form, lack Phe-tRNAPhe binding activity and further that 70S ribosomes containing such reconstituted 30S subunits have substantially diminished binding activity to both the A and P sites, as differentiated through use of tetracycline. Suitable control experiments strongly indicate that this loss of activity is a direct consequence of puromycin photoincorporation.
D000345:Affinity Labels; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism*; D010449:Peptide Mapping; D011691:Puromycin / Q000378:metabolism / Q000494:pharmacology*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D014316:Tritium
D000345:Affinity Labels; D012346:RNA, Transfer, Amino Acyl; D012269:Ribosomal Proteins; C039747:ribosomal protein S14; D014316:Tritium; D011691:Puromycin