Monoclonal antibodies to Escherichia coli ribosomal proteins L9 and L10. Effects on ribosome function and localization of L9 on the surface of the 50 S ribosomal subunit.
Monoclonal antibodies against Escherichia coli ribosomal proteins L9 and L10 were obtained and their specificity confirmed by Western blot analysis of total ribosomal protein. This was particularly important for the L9 antibody, since the immunizing antigen mixture contained predominantly L11. Each antibody recognized both 70 S ribosomes and 50 S subunits. Affinity-purified antibodies were tested for their effect on in vitro assays of ribosome function. Anti-L10 and anti-L9 inhibited poly(U)-directed polyphenylalanine synthesis almost completely. The antibodies had no effect on subunit association or dissociation and neither antibody inhibited peptidyltransferase activity. Both antibodies inhibited the binding of the ternary complex that consisted of aminoacyl-tRNA, guanylyl beta, gamma-methylenediphosphonate, and elongation factor Tu, and the binding of elongation factor G to the ribosome. The intact antibodies were more potent inhibitors than the Fab fragments. In contrast to the previously established location of L10 at the base of the L7/L12 stalk near the factor-binding site, the site of anti-L9 binding to 50 S subunits was shown by immune electron microscopy to be on the L1 lateral protuberance opposite the L7/L12 stalk as viewed in the quasisymmetric projection. The inhibition of factor binding by both antibodies, although consistent with established properties of L10 in the ribosome, suggests a long range effect on subunit structure that is triggered by the binding of anti-L9.
Nag
B
B
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Akella
S S
SS
Cann
P A
PA
Tewari
D S
DS
Glitz
D G
DG
Traut
R R
RR
D000818:Animals; D000911:Antibodies, Monoclonal; D004926:Escherichia coli / Q000378:metabolism*; D005260:Female; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008854:Microscopy, Electron; D008961:Models, Structural; D010452:Peptide Biosynthesis; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010455:Peptides; D011072:Poly U; D000080882:Ribosomal Protein L10; D012269:Ribosomal Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D010455:Peptides; D012269:Ribosomal Proteins; C000630690:Rpl10 protein, mouse; C049650:ribosomal protein L9; D011072:Poly U; C012001:polyphenylalanine; D010444:Peptide Elongation Factor Tu