INCORPORATION OF DINITROPHENYL PROTEIN L23 INTO TOTALLY RECONSTITUTEDESCHERICHIA-COLI 50-S RIBOSOMAL-SUBUNITS AND ITS LOCALIZATION AT 2 SITES BY IMMUNE ELECTRON-MICROSCOPY

Escherichia coli ribosomal protein L23 was derivatized with [H-3]2,4-d initrofluorobenzene both at the N terminus and at internal lysines. Di nitrophenyl-L23 (DNP-L23) was taken up into 50 S subunits from a recon stitution mixture containing rRNA and total 50 S protein depleted in L 23. Unmodified L23 competed with DNP-L23 for uptake, indicating that e ach protein form bound in an identical or similar position within the subunit. Modified L23, incorporated at a level of 0.7 or 0.4 DNP group s per 50 S, was localized by electron microscopy of subunits complexed with antibodies to dinitrophenol. Antibodies were seen at two major s ites with almost equal frequency. One site is beside the central protu berance, in a region previously identified as the peptidyltransferase center. The second location is at the base of the subunit, in the area of the exit site from which the growing peptide leaves the ribosome. Models derived from image reconstruction show hollows or canyons in th e subunit and a tunnel that Links the transferase and exit sites. Our results indicate that L23 is at the subunit interior, with separate el ements of the protein at the subunit surface at or near both ends of t his tunnel.