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Reconstitution of Escherichia coli 50S ribosomal subunits containing puromycin-modified L23: functional consequences
1Department of Chemistry, University of Pennsylvania, Philadelphia 19104.
Abstract:
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].
Insights
Photoaffinity labeling of Escherichia coli ribosomal protein L23 with puromycin demonstrates its proximity to the peptidyl transferase center. Modified L23 interferes with tRNA binding, suggesting a role in ribosomal function.
Area of Science:
- Molecular Biology
- Ribosome Biochemistry
- Protein-Nucleic Acid Interactions
Background:
- Previous studies identified protein L23 of Escherichia coli ribosomes as a primary target for photoaffinity labeling by puromycin and p-azidopuromycin.
- Protein L23 is extensively labeled, indicating its accessibility and potential functional significance within the ribosome.
Purpose of the Study:
- To investigate the functional consequences of modifying ribosomal protein L23 with puromycin.
- To determine the precise location of protein L23 relative to the peptidyl transferase center and its role in tRNA binding.
Main Methods:
- Site-specific photoaffinity labeling of protein L23 within intact 70S ribosomes using puromycin.
- Separation of modified L23 (puromycin-L23) from unmodified L23 using reverse-phase high-performance liquid chromatography (RP-HPLC).
- Reconstitution of 50S ribosomal subunits using puromycin-L23 and assessment of peptidyl transferase activity and tRNA binding.
Main Results:
- Puromycin-L23 was successfully incorporated into reconstituted 50S subunits at a maximum of 0.5 copies per subunit.
- Reconstituted subunits with puromycin-L23 retained significant peptidyl transferase activity but showed reduced mRNA-dependent tRNA binding (50-60%).
- These findings suggest L23 is close to, but not directly within, the peptidyl transferase center.
Conclusions:
- Protein L23 is located near the peptidyl transferase center of the Escherichia coli ribosome.
- Modification of L23 with puromycin impairs tRNA binding, indicating L23's involvement in this ribosomal function.
- The results support models placing L23 in proximity to the peptidyl transferase center, though discrepancies with some structural data remain.