Reconstitution of Escherichia coli 50S ribosomal subunits containing puromycin-modified L23: functional consequences.
In previous work we have shown that both puromycin [Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274] and p-azidopuromycin [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Coooperman, B. S. (1982) Biochemistry 21, 3809-3817] site specifically photoaffinity label protein L23 to the highest extent of any Escherichia coli ribosomal protein. In this work we demonstrate that L23 that has been photoaffinity labeled within a 70S ribosome by puromycin (puromycin-L23) can be separated from unmodified L23 by reverse-phase high-performance liquid chromatography (RP-HPLC) and further that puromycin-L23 can reconstitute into 50S subunits when added in place of unmodified L23 to a reconstitution mixture containing the other 50S components in unmodified form. We have achieved a maximum incorporation of 0.5 puromycin-L23 per reconstituted 50S subunit. As compared with reconstituted 50S subunits either containing unmodified L23 or lacking L23, reconstituted 50S subunits containing 0.4-0.5 puromycin-L23 retain virtually all (albeit low) peptidyl transferase activity but only 50-60% of mRNA-dependent tRNA binding stimulation activity. We conclude that although L23 is not directly at the peptidyl transferase center, it is sufficiently close that puromycin-L23 can interfere with tRNA binding. This conclusion is consistent with a number of other experiments placing L23 close to the peptidyl transferase center but is difficult to reconcile with immunoelectron microscopy results placing L23 near the base of the 50S subunit on the side facing away from the 30S subunit [Hackl, W., & Stöffler-Meilicke, M. (1988) Eur. J. Biochem. 174, 431-435].
Department of Chemistry, University of Pennsylvania, Philadelphia 19104.
C J
CJ
Weitzmann
B S
BS
Cooperman
D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D011485:Protein Binding; D011691:Puromycin / Q000378:metabolism* / Q000494:pharmacology; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012269:Ribosomal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure
D029968:Escherichia coli Proteins; D012360:RNA, Transfer, Phe; D012269:Ribosomal Proteins; C043816:ribosomal protein L23, E coli; D011691:Puromycin