Synthetic Biology of Small RNAs and Riboswitches

Abstract

In bacteria and archaea, small RNAs (sRNAs) regulate complex networks through antisense interactions with target mRNAs in trans, and riboswitches regulate gene expression in cis based on the ability to bind small-molecule ligands. Although our understanding and characterization of these two important regulatory RNA classes is far from complete, these RNA-based mechanisms have proven useful for a wide variety of synthetic biology applications. Besides classic and contemporary applications in the realm of metabolic engineering and orthogonal gene control, this review also covers newer applications of regulatory RNAs as biosensors, logic gates, and tools to determine RNA-RNA interactions. A separate section focuses on critical insights gained and challenges posed by fundamental studies of sRNAs and riboswitches that should aid future development of synthetic regulatory RNAs.

FIGURE 1

Timeline of sRNA and riboswitch discovery, including relevant technological advances that aided identification and verification of regulatory RNAs. The development of high-throughput, deep-sequencing techniques in particular has led to an explosion of sRNA and riboswitch discovery. However, although identification of sRNAs and riboswitches has rapidly expanded, verification of function still lags behind.

FIGURE 2

General function of sRNAs (A to D) and riboswitches (a to f). sRNAs regulate gene expression in trans through several functions enacted by antisense interactions, including transcription attenuation/enhancement through interactions with the RNA polymerase (A), inhibition of protein or ribosome binding either indirectly (B) or directly (C), and sequestration of protein factors (such as CsrA) (D). Riboswitches regulate gene expression in cis through a ligand-induced conformational change in the expression platform. The resulting gene expression consequences include Rho-dependent/independent transcription termination (a, b), transcription antitermination (c), translation activation (d), translation inhibition (e), and mRNA degradation (f).

FIGURE 3

Examples of applications of sRNAs and riboswitches. Applications of these regulatory RNAs are rooted in their unique functional characteristics (antisense interactions for sRNA and ligand binding for riboswitches). Recent applications of these systems have begun to interweave these mechanisms to provide more complex engineering strategies.