Novel amino acid activating ribozyme KK33-1 with clues to its functional architecture

The first step in contemporary protein synthesis is the activation of amino acids by aminoacyl-tRNA synthetases, which form aminoacyl adenylates from ATP and amino acids. Previously, an RNA called KK13 was shown to catalyze amino acid activation by forming a bond between an amino acid and the α-phosphate of the 5'-triphosphate of its own RNA, analogous to the bond formed in aminoacyl adenylates. In the present study, we describe a putative novel RNA with catalytic activity, identified among previously uncharacterized sequences. The RNA, KK33-1 (114 nucleotides), exhibited activity comparable to that of a previously characterized ribozyme, KK13, as judged by a malachite green assay detecting pyrophosphate released from the 5'-triphosphate of the RNA. Guided by predicted secondary structure, truncation experiments indicated that certain smaller variants of KK33-1 can retain amino acid activation activity. Although attention was drawn to a CAAC sequence common to KK13 and KK33-1, which was positioned near the putative reaction site according to tertiary structure prediction, a mutational analysis revealed that this motif is not essential for catalysis. Together, the behaviors of KK33-1 and its mutants provide insights into the structural features supporting amino acid activation, refining our understanding of the ribozyme's functional architecture.