Rapid and Simple Ribozymic Aminoacylation Using Three Conserved Nucleotides
Selection-amplification finds new RNA enzymes (ribozymes) among randomized RNAs with flanking unvaried sequences (primer complements). Precise removal of 3?-primer before reaction selected aminoacylation from PheAMP in three cycles, yielding active RNAs (kcat = 12?20 min?1) using only three conserved nucleotides, acting independently of divalent ions. This unusually simple RNA active site encouraged study of the reaction via molecular mechanics-based free energy minimization. On this basis, we suggest a chemical path for RNA-catalyzed transaminoacylation. Site modeling also predicted new features, l-stereoselectivity, 2?-regioselectivity, independence of amino acid side chain, and phosphorylated activating group, that were subsequently verified. The same selection also showed that RNA aminoacylation from adenylate is simpler than from CoA thioester, potentially rationalizing translational activation by adenylates. The simplicity of this active site suggests a general route to small ribozymes.