Probing the functional role and localization of Escherichia coli ribosomal protein L16 with a monoclonal antibody.
A monoclonal antibody specific for Escherichia coli ribosomal protein L16 was prepared to test its effects on ribosome function and to locate L16 by immunoelectron microscopy. The antibody recognized L16 in 50 S subunits, but not in 70 S ribosomes. It inhibited association of ribosomal subunits at 10 mM Mg2+, but not at 15 mM Mg2+. Poly(U)-directed polyphenylalanine synthesis and peptidyltransferase activities were completely inhibited when the L16 antibody was bound to 50 S subunits at a molar ratio of 1. There was no inhibitory effect on the binding of elongation factors or on the associated GTPase activities. Fab fragments of the antibody gave the same result as the intact antibody. Chemical modification of the single histidine (His13) by diethyl pyrocarbonate destroyed antibody binding. Electron microscopy of negatively stained antibody subunit complexes showed antibody binding beside the central protuberance of the 50 S particle on the side away from the L7/L12 stalk and on or near the interface between the two subunits. This site of antibody binding is fully consistent with its biochemical effects that indicate that protein L16 is essential for the peptidyltransferase activity activity of protein biosynthesis and is at or near the subunit interface.
Nag
B
B
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Glitz
D G
DG
Tewari
D S
DS
Traut
R R
RR
D000911:Antibodies, Monoclonal; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D007140:Immunoglobulin Fab Fragments; D008854:Microscopy, Electron; D008961:Models, Structural; D010452:Peptide Biosynthesis; D010455:Peptides; D011072:Poly U / Q000378:metabolism; D012269:Ribosomal Proteins / Q000032:analysis / Q000378:metabolism* / Q000648:ultrastructure; D012270:Ribosomes / Q000378:metabolism / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D010455:Peptides; D012269:Ribosomal Proteins; C027937:ribosomal protein L16; D011072:Poly U; C012001:polyphenylalanine