Incorporation of Dinitrophenyl Protein L23 into Totally Reconstituted Escherichia coli 50 S Ribosomal Subunits and Its Localization at Two Sites by Immune Electron Microscopy

Abstract

Escherichia coli ribosomal protein L23 was derivatized with [ 3 H]2,4-dinitrofluorobenzene both at the N terminus and at internal lysines. Dinitrophenyl-L23 (DNP-L23) was taken up into 50 S subunits from a reconstitution mixture containing rRNA and total 50 S protein depleted in L23. Unmodified L23 competed with DNP-L23 for uptake, indicating that each 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 groups per 50 S, was localized by electron microscopy of subunits complexed with antibodies to dinitrophenol. Antibodies were seen at two major sites with almost equal frequency. One site is beside the central protuberance, 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 the subunit and a tunnel that links the transferase and exit sites. Our results indicate that L23 is at the subunit interior, with separate elements of the protein at the subunit surface at or near both ends of this tunnel.Determination of the positions of proteins within the 30 S and 50 S ribosomal subunits has been a major goal of studies directed toward the elucidation of the structure and function of the Escherichia coli ribosome. The technique of immune electron microscopy (IEM), 1 the visualization in electron micrographs of complexes formed between ribosomal subunits and specific antibodies, has been especially useful for this purpose (1, 2). However, in the case of protein L23, application of IEM has yielded controversial results. The object of the present work is to resolve this controversy.E. coli ribosomal protein L23 is a primary binding protein; it interacts directly with ribosomal RNA (3) and plays a significant role in the assembly of the large ribosomal subunit (4). The protein has been linked to the ribosomal peptidyltransferase center in several ways. First, photoaffinity labeling of 70 S ribosomes with either puromycin (5-7) or p-azidopuromycin (8, 9), each of which inhibits protein synthesis by acting as a peptide acceptor in the transferase reaction, led to photoincorporation into L23 as the major site of protein labeling. Second, antibodies recognizing the N 6 ,N 6 -dimethyladenosine moiety of puromycin bound to labeled 50 S subunits in a region generally agreed to include the peptidyltransferase center, i.e. between the central protuberance and the site of protein L1, near the 30 S:50 S interface (10, 11). Third, chemical cross-linking and related studies showed L23 to neighbor other proteins (L2, L15, L16, and L27) that have been placed at or near the peptidyltransferase center (12). Finally, although peptidyltransferase activity is not altered in reconstituted 50 S subunits either lacking L23 or including puromycin-modified L23 in place of L23, the latter do show reduced aminoacyl-tRNA binding (6), which suggests proximity to t...

Reconstitution of Functional 50S Ribosomes from in Vitro Transcripts of Bacillus stearothermophilus 23S rRNA

“…These reconstitution systems are a powerful tool for the analysis of the structure, function, and assembly of the 50S subunit ( − ). An in vitro reconstitution system dependent on in vitro-generated transcripts of 23S rRNA would provide an improved approach to in vitro genetics of 23S rRNA.…”