Cooperative binding of 3′-fragments of transfer ribonucleic acid to the peptidyltransferase center of Escherichia coli ribosomes

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Abstract

The binding of substrates to the A-site half (A′) and the P-site half (P′) of the peptidyltransferase center was measured by means of equilibrium dialysis. The tRNA fragments C-A-C-C-A-Leu and C-A-C-C-A-(N-acetyl)Leu were used as A′-site and P′-site substrates, respectively. The A′- and P′-substrates bound well to 50 S in contrast to 30 S subunits; significant binding to 23 S and 16 S RNA was also found. The binding of the P′-site substrate to 23 S RNA and 50 S subunits was very similar at various Mg2+ and K+ concentrations, indicating that the 23 S RNA is probably directly involved in the binding of the 3′-end of the peptidyl-tRNA. Cooperative effects at the peptidyltransferase center were found using chloramphenicol and deacylated tRNA as competitors, which completely inhibited the substrate binding to one site whilst drastically stimulating binding to the other. Chloramphenicol inhibited the binding of the A′-site substrate C-A-C-C-A-Leu, whereas the binding of the corresponding P′-site substrate was stimulated. In contrast, deacylated tRNA blocked the binding of the P′-site substrate, but stimulated the corresponding A′-site binding. Similarly, the trinucleotide Cp,CpA inhibited binding of the P′-site substrate (showing complete inhibition at 70 μm) whereas binding of the A′-site substrate was slightly stimulated at concentrations below 70 μm.

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