A New Role for the CYT-18 N-Terminus and Three-Dimensional DNA Crystals as Vehicles for Biocatalysis

Abstract

The bifunctional Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (N. crassa mt TyrRS; CYT-18 protein) promotes the splicing of multiple group I introns by stabilizing the catalytically active intron structures. CYT-18, and mt TyrRS's from related fungal species, have evolved to promote group I intron splicing partly by accumulation of three N-terminal domain insertions that create a structure-stabilizing scaffold for critical tertiary interactions between the two major group I intron domains. The primarily alpha-helical N-terminal insertion, H0, contributes to protein stability and is necessary for splicing the N. crassa ND1 intron, but is dispensable for splicing the N. crassa mt LSU intron. Herein, I show CYT-18 with a complete H0 deletion retains residual ND1 intron splicing activity and addition of the missing N-terminus in trans restores a significant portion of its splicing activity. This peptide complementation assay revealed important characteristics of the CYT-18/group I intron interaction including the stoichiometry of H0 in intron splicing and the importance of specific H0 residues. Evaluation of truncated H0 peptides in this assay also suggests a previously unknown structural role of the first five N-terminal residues of CYT-18. These residues interact directly with another splicing insertion, making H0 a central structural element responsible for connecting all three N-terminal splicing insertions.

Transitioning to a separate study, I have demonstrated that enzymes retain catalytic activity when captured in the solvent channels of three-dimensional (3D) DNA crystals. Using RNase A as a model enzyme system this work shows that crystals infused with enzyme can cleave a fluorescent dinucleotide substrate with similar kinetic restrictions as other immobilized enzyme systems, mainly limited by diffusion of substrate. This new vehicle for immobilized enzymes, created entirely from biomolecules, provides a platform for developing modular solid-state catalysts that could be both biocompatible and biodegradable.