70-S Ribosomes of Escherichia coli Have an Additional Site for Deacylated tRNA Binding

Abstract

Escherichia coli 70-S ribosomes contain a third site for tRNA binding, additional to the A and P sites. This conclusion is based on several findings. Direct measurements showed that in the presence of poly(U), when both A and P sites are occupied by Ac[¹⁴C]Phe-tRNA^Phe, ribosomes are capable of binding additionally deacylated non-cognate [³H]tRNA. If ribosomes in the preparation are active enough, the total binding of labeled ligands amounted to 2.5 mol/mol ribosomes. In the absence of poly(U), when the A site can not bind, the P site and the ‘additional’ site can be filled simultaneously with Ac[³C]Phe-tRNA^Phe and deacylated [³H]tRNA, or with [³H]tRNA alone; the total binding exceeds in this case 1.5 mol/mol ribosomes. The binding at the ‘additional’ site is not sensitive to the template. [³H]tRNA bound there is able to exchange rapidly for unlabeled tRNA in solution. Deacylated tRNA is prefered to the aminoacylated one. The binding of AcPhe-tRNA^Phe was not observed there at all. The 3′-end adenosine is essential for the affinity. The function of the ‘additional’ site is not known, but its existence has to be considered when tRNA · ribosome complexes are studied.