Mechanism of codon-anticodon interaction in ribosomes. Direct functional evidence that isolated 30S subunits contain two Codon-specific binding sites for transfer RNA

Abstract

308 subunits were isolated capable to bind simultaneously two molecules of Phe-tRNA^Phe (or N-Acetyl-Phe-tRNA^Phe), both poly(U) dependent. The site with higher affinity to was identified as P site. tRNA binding to this site was not inhibited by low concentrations of tetracycline (2×10⁻⁵M) and, on the other hand, N-Acetyl-Phe-tRNA^Phe, initially prebound to the 30S·poly(U) complex in the presence of tetraoycline, reacted with puromycin quantitatively after addition of 50S subunits. The site with lower affinity to tRNA revealed features of the A site: tetracycline fully inhibited the binding of both Phe-tRNA^Phe and N-Acetyl-Phe-tRNA^Phe. Binding of two molecules of Phe-tRNA^Phe to the 30S·poly(U) complex followed by the addition of 50s subunits resulted in the formation of (Phe)₂-tRNA^Phe in 75–90% of the reassociated 70S ribosomes. These results prove that isolated 30S subunits contain two physically distinct centers for the binding of specific aminoacyl- (or peptidyl-) tRNA. Addition of 50S subunits results in the formatfon of whole 70S ribosomes with usual donor and acceptor sites.