Incorporation of dinitrophenyl protein L23 into totally reconstituted Escherichia coli 50 S ribosomal subunits and its localization at two sites by immune electron microscopy.
Escherichia coli ribosomal protein L23 was derivatized with [3H]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.
Montesano-Roditis
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Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095-1737, USA.
Glitz
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DG
Perrault
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AR
Cooperman
B S
BS
D000906:Antibodies / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004139:Dinitrofluorobenzene; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D008854:Microscopy, Electron; D008958:Models, Molecular; D012329:RNA, Bacterial / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000378:metabolism
D000906:Antibodies; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; C043816:ribosomal protein L23, E coli; D004139:Dinitrofluorobenzene