Mechanism of codon—anticodon interaction in ribosomes

According to current concepts in protein biosyn: thesis, aminoacyl-tRNA is never placed at the donor (D) site of the ribosome during elongation, because this site, at that time, is always occupied by nascent peptidyl-tRNA. But in model systems, containing free ribosomes charged with mRNA, aminoacyl-tRNA can bind both at D and A (acceptor) sites. It was shown earlier that at low [Mg’+] (5-6 mM) the D site was preferred, but at higher [Mg’+] most of the bound aminoacyl-tRNA was found at the A site [ 1,2]. This was true also for peptidyl-tRNA [3,4]. However in [5], enriched Phe-tRNAPhe bound initially at the D site both at low (10 mM) and high (30 mM) [Mg'+] . Moderate concentrations of cognate tRNAPhe, but not noncognate tRNAs, were found to be strong competitors for D-site binding [5]. The data mentioned are somewhat contradictory. Thus, we reexamined the problem of Mg2+-dependent aminoacyl-tRNA distribution between the A and D sites of 70 S . poly(U) complex. Using ribosomes with fully active sites, we were able to show that purified Phe-tRNAPhe has a higher affinity towards the D site at all [Mg2’] studied (5-20 mM); at excess of ribosomes Phe-tRNAPhe binds exclusively at the D site. However, if nonenriched preparations of Phe-tRNA were used, the latter was found preferentially in the A site (up to 80%, at 20 mM Mg2+). Obviously, a high excess of natural mixture of non-cognate deacylated tRNAs is also a strong competitor for D-site binding, at least at high [Mg”]. This finding allowed us to measure the association