Fitness landscape of a dynamic RNA structure
In this study, we examined the fitness landscape of a dynamic RNA structure using deep mutational scanning (DMS) experiments. Using a derivative of the group I intron from Tetrahymena thermophila, we measured fitness for almost all intron genotypes that vary at N2-N5 and N18-N21, two sub-regions that base-pair and form the P1 extension (P1ex) helix adjacent to the 5' splicing site. Following the cleavage at the 5' splice site during the self-splicing reaction, P1ex needs to dissociate to allow formation of a second helix (P10), where the N18-N21 sub-region pairs with bases at the 5' end of the 3' exon. To measure molecular fitness and characterize epistatic interactions, we used a developed heterologous reporter system where the intron is embedded in a kanamycin nucleotidyltransferase (knt) gene, placed on a plasmid and transformed into E. coli. This system couples self-splicing activity to fitness as intron removal is required for the reconstitution of the knt open reading frame whose translation enables growth in the presence of kanamycin. The N2-N5 and N18-N21 sub-regions of the intron were subjected to saturation mutagenesis to generate all possible genotypes, which were then pooled and transformed into Escherichia coli. Six independent transformations were performed, representing six biological replicates. Each biological replicate was split evenly into four aliquots, which were spread on agar plates that did or did not contain kanamycin and incubated at either 30C or 37C. After overnight incubation, genotype frequencies of all intron mutants were assayed via high-throughput amplicon sequencing. Our results demonstrate the asymmetric and pleiotropic constraints imposed on the two sub-regions of the intron, and overall capture fitness effects arising from multiple transient conformational states of the intron.