Enhancement of the phenylalanyl-oligonucleotide binding to the peptidyl recognition center of ribosomal peptidyltransferase and inhibition of the chloramphenicol binding to ribosomes

Abstract

The binding of C-A-C-C-A(Phe) to ribosomes was critically affected by the concentration of ethanol and C-A-C-C-A(Phe) in the reaction mixture. In the presence of sparsomycin, the binding was inhibited at a low ethanol concentration, whereas it was stimulated at a moderately high ethanol concentration. At a moderately high concentration of ethanol, plots of the initial velocity of C-A-C-C-A(Phe) binding to ribosomes against C-A-C-C-A(Phe) concentration exhibited a sigmoidal saturation curve; while at low ethanol concentration, it gave a hyperbolic saturation curve. C-A-C-C-A(Phe) could not react with puromycin if a low level of C-A-C-C-A(Phe) and a low concentration of ethanol were included in the reaction mixture, while it did react with puromycin to form phenylalanyl-puromycin when a high level of C-A-C-C-A(Phe) and a high concentration of ethanol were used. In the presence of a moderately high concentration of ethanol, the binding of chloramphenicol to ribosomes was markedly inhibited by C-A-C-C-A(Phe) or C-A-C-C-A(Ac-Phe), while sparsomycin enhanced the inhibition. In the absence of ethanol, the phenylalanyl-oligonucleotides slightly affected the chloramphenicol binding to ribosomes. On the basis of these findings, it was assumed that C-A-C-C-A(Phe) could bind to the peptidyl recognition center as well as the aminoacyl recognition center of ribosomes, and that chloramphenicol could bind to the ribosomes at or very close to the peptidyl recognition center of the peptidyl transferase.