A NOVEL CELL-FREE SYSTEM FOR PEPTIDE-SYNTHESIS DRIVEN BY PYRIDINE

Previously we demonstrated that ribosomes can synthesize polypeptides in the presence of high concentrations (40-60%) of pyridine without an y protein factors, Here we analyze additional ribosomal parameters in 60% pyridine using Escherichia coli ribosomes, Ribosomal subunits once exposed to pyridine failed to re-associate to 70S ribosomes in aqueou s buffer systems even in the presence of 20 mM Mg2+, whereas they form ed 70S complexes in the presence of 60% pyridine, Two-dimensional gel electrophoresis of ribosomal proteins revealed that some proteins loca ted at the protuberances of the large subunit, e, g. L7/L2 and L11 for ming the elongation factor-binding domain, were released in the pyridi ne system. The aminoglycoside neomycin, a strong inhibitor of the ribo somal (factor-independent) translocation reaction, completely blocked poly(Phe) synthesis and translocation activities in the pyridine syste m, whereas these activities were not affected at all by gypsophilin, a ribotoxin that inhibits factor-dependent translocation, Another inhib itor of the ribosomal translocation, thiostrepton, had no effect conce rning the two activites, which is consistent with the fact that this a ntibiotic requires L11 for its binding to the ribosome, These results suggest that the ribosomes can perform a translocation reaction in the pyridine system, but in a factor-independent (spontaneous) manner.