High-throughput functional profiling and evolutionary covariation analysis of entire riboswitch sequences.

Riboswitches are useful models for revealing how some RNA molecules undergo dynamic rearrangements of their structures to perform cellular functions. A great deal is known about riboswitch aptamer domains through sequence covariation analysis, which has been difficult to apply to expression platforms given their large sequence diversity. Here, we develop an approach to generate covariation models for entire riboswitch sequences including the aptamer domain and the expression platform. The method consists of bioinformatically extending aptamer domains to include downstream sequences and filtering these sequences using either computational or high-throughput experimental approaches to identify those that include bacterial intrinsic terminators. Filtered sequences are then used to generate covariation models. We developed this approach in the context of the fluoride riboswitch, characterizing 1901 fluoride riboswitch sequences using high-throughput in vitro transcription followed by next-generation sequencing, and generating a covarion model consistent with its mechanism. We then developed covariation models of the ZTP, lysine, and TPP riboswitches and find covariation support for previously published mechanisms. Our method represents a new approach to characterizing large numbers of riboswitch sequences and to generate covariation models of complete riboswitches, which should expand our understanding of riboswitch mechanisms and the evolution of RNA structure dynamics.