title: The number of copies of ribosome-bound proteins L7 and L12 required for protein synthesis activity.
abstract: Poly(U)-dependent poly(Phe) synthesis and elongation factor G (EF-G)-dependent GTPase activity were used to study the partial reconstitution of L7/L12-deficient ribosomes with proteins L7/L12 and fluorescent conjugates. Seventy-five per cent of these activities are restored when unmodified L7/L12 dimer is added to L7/L12-deficient cores at a ratio of 1:1. Various covalent fluorescent conjugates of L7/L12 bind to these cores about as well as unmodified protein. A fluorescein-5-isothiocyanate derivative of L12 shows almost no functional activity when bound. However, mixed reconstitutes of this conjugate and unmodified L12 have 75% functional activity when half the protein is unmodified. These results can be explained by a model in which there are two independent binding sites on the ribosome for two dimers of L7/L12. The binding of dimers to ribosomes is totally random and complete; the particle is 100% active so long as it has one active dimer bound to either one of the two sites. However, more complex models cannot be ruled out. An 5-(iodoacetamidoethyl)-aminonaphthalene-1-sulfonic acid (IAEDANS) derivative of L7 is labeled semispecifically at the COOH terminus. This conjugate shows partial functional activity. When assay results are analyzed using the above model, it appears that the specific COOH-terminal modification has no effect on activity. However, all but a small fraction of the nonspecific IAEDANS modifications lead to inactivation.
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism*; D007460:Iodoacetamide / Q000031:analogs & derivatives; D007700:Kinetics; D009282:Naphthalenesulfonates; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011072:Poly U; D014176:Protein Biosynthesis; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism
chemical_list: D009282:Naphthalenesulfonates; D010445:Peptide Elongation Factors; D010448:Peptide Initiation Factors; D012269:Ribosomal Proteins; D011072:Poly U; C006421:1,5-I-AEDANS; C016700:ribosomal protein L7-L12; D010744:Phosphoric Monoester Hydrolases; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D007460:Iodoacetamide