Two monoclonal antibodies against Escherichia coli ribosomal protein L2 distinguish different locations for their respective epitopes in intact ribosomes.
Two monoclonal antibodies raised against intact Escherichia coli ribosomal protein L2 were isolated, affinity-purified, and characterized. One of the antibodies (Ab 5-186) recognizes an epitope within residues 5-186, and the other (Ab 187-272) recognizes an epitope within residues 182-272. Both antibodies strongly inhibit in vitro polyphenylalanine synthesis when they are first allowed to bind to 50 S subunits prior addition of 30 S subunits. However, only Ab 187-272 is inhibitory when added to preformed 70 S ribosomes. Ab 5-186 binds to 50 S subunits but not to 70 S ribosomes. Ab 187-272 does not cause dissociation of 70 S ribosomes under the ionic conditions of the assay for polyphenylalanine synthesis (15 mM magnesium), although at 10 mM magnesium it does cause dissociation. Both antibodies inhibit the reassociation of 50 S with 30 S subunits. Both antibodies strongly inhibit peptidyltransferase activity. The two antibodies differ in their effects on interactions with elongation factors Tu (EF-Tu) and G (EF-G). Neither antibody significantly inhibits EF-G-dependent GTPase activity, nor the binding of EF-G when the antibodies are incubated with 50 S subunits; however, Ab 187-272 causes a decrease in the binding of EF-Tu X aminoacyl-tRNA X GTP ternary complex and of EF-Tu-dependent GTPase when it is incubated with 70 S ribosomes. The Fab fragments of both antibodies had effects similar to the intact antibodies. The results show that monoclonal antibodies can be used to discriminate different regions of L2 and that EF-Tu and EF-G do not have identical ribosomal binding sites.
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000032:analysis / Q000276:immunology*; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D046911:Macromolecular Substances; D010452:Peptide Biosynthesis; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010455:Peptides; D010458:Peptidyl Transferases / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D012269:Ribosomal Proteins / Q000276:immunology*; D012270:Ribosomes / Q000032:analysis*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D010455:Peptides; D012269:Ribosomal Proteins; C029466:ribosomal protein L2; D011072:Poly U; C012001:polyphenylalanine; D010458:Peptidyl Transferases; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu