Abstract

The minimal ribozyme drz-Mtgn-1 when compared to other similar minimal ribozymes derived from the same deep sequenced metagenomic human diarrheal sample appeared to have a decrease in catalytic activity beyond 1mM concentration of Mg²⁺. Intriguingly, this behavior vastly differs from the standard activity-metal curve trends previously observed in HDV and HDV-like ribozymes and suggests that the decrease in catalytic activity is due to a conformational change at high divalent metal concentrations. The nucleic acid sequence of drz-Mtgn-1 was successfully inserted into the pUC19 plasmid and cloned by DH5α Escherichia coli cells. Following plasmid extraction, the cloned plasmid containing the drz-Mtgn-1 sequence was transcribed under optimal conditions. The purified drz-Mtgn-1 ribozyme was refolded at both 1 mM and 10 mM concentrations of Mg²⁺ and subject to hanging-drop crystallization. Several crystals were produced under both 1 mM and 10 mM Mg²⁺ concentrations. By subjecting these crystals to x-ray crystallography, the proposed dual conformations of drz-Mtgn-1 shall be examined by analyzing its unique molecular structure.