Engineering ligand-responsive gene-control elements: lessons learned from natural riboswitches

Abstract

In the last two decades, remarkable advances have been made in the development of technologies used to engineer new aptamers and ribozymes. This has encouraged interest among researchers who seek to create new types of gene-control systems that can be made to respond specifically to small-molecule signals. Validation of the fact that RNA molecules can exhibit the characteristics needed to serve as precision genetic switches has come from the discovery of numerous classes of natural ligand-sensing RNAs called riboswitches. Although a great deal of progress has been made toward engineering useful designer riboswitches, considerable advances are needed before the performance characteristics of these RNAs match those of protein systems that have been co-opted to regulate gene expression. In this review, we will evaluate the potential for engineered RNAs to regulate gene expression and lay out possible paths to designer riboswitches based on currently available technologies. Furthermore, we will discuss some technical advances that would empower RNA engineers who seek to make routine the production of designer riboswitches that can function in eukaryotes.

Figure 1

Natural Riboswitch Locations and Mechanisms. (a) Transcription regulation based on a guanine-sensing riboswitch. When guanine (G) binds, the folding pathway favors the formation of a terminator stem at the expense of antiterminator stem formation. This architecture yields ‘off’ switch function because guanine causes transcription to terminate before the coding region of the mRNA is synthesized. Less common are examples of ‘on’ switch function where ligand binding favors antiterminator stem formation, as is observed with a related adenine-sensing riboswitch. (b) Translational regulation based on a TPP riboswitch. In the absence of the riboswitch ligand, expression of the down stream gene is permitted because translation can be initiated at the ribosomal binding site (RBS). However, in the presence of the ligand, the RNA adopts an alternate conformation that does not permit translation to be initiated at the RBS. (c) Metabolite-triggered ribozyme regulation of gene expression by GlcN6P riboswitches. When the ribozyme cofactor GlcN6P is low in concentration, the ribozyme does not undergo efficient self-cleavage and the stable mRNA can be translated. When GlcN6P is bound by the RNA, the ribozyme undergoes efficient self-cleavage, and the 3′ cleavage fragment including the ORF is rapidly degraded by a nuclease. (d) Control of alternative splicing by a TPP-sensing riboswitch in eukaryotes. When TPP concentrations are low, nucleotides from the unoccupied aptamer base pair near the second 5′ splice site, forcing the spliceosome to use the first 5′ splice site. When TPP is bound, the nucleotides formerly blocking the splice site are now involved in binding the ligand. This allows the spliceosome to choose the second 5′ splice site to yield an alternatively spliced mRNA. In fungi, ligand binding yields an alternatively spliced mRNA lacking upstream open reading frames (uORFs) that otherwise would decoy the ribosome from initiating translation at the main ORF. (e) Eubacterial riboswitch placement. Most riboswitches found in eubacteria are present within the 5′ untranslated regions (UTR) of mRNAs and directly control expression of a downstream open reading frame (ORF). There is bioinformatics evidence that at least one riboswitch controls the expression of an antisense RNA to regulate protein expression from a separate mRNA indirectly. (f) Eukaryotic riboswitch placement. Riboswitches have been found in introns located within the 5′ UTRs, coding regions, and 3′ UTRs of eukaryotic mRNAs.

Figure 2

Aptamer-based synthetic riboswitches. (a) Aptamer-mediated inhibition of ribosomal scanning in S. cerevisiae. Ligand binding by the aptamer stabilizes a structure within the 5′ UTR that precludes the ribosome from reaching the AUG start codon. Black lines represent UTRs, green line represents the start codon, dark blue line identifies the ORF, and the light blue line represents the aptamer domain. (b) Aptamer mediated inhibition of mRNA splicing in S. cerevisiae. Ligand binding stabilizes a structure that sequesters the 5′ splice site (5′ SS), which precludes efficient splicing. BP and 3′ SS designate the branch point and the 3′ splice site, respectively. Additional annotations are as described in a. (c) Aptamer mediated inhibition of ribosome binding in E. coli. The absence of ligand binding allows the aptamer sequence to base pair with the ribosome binding site (RBS), which inhibits ribosome binding. Annotations are as described in a.

Figure 3

Functional parameters for aptamer- and allosteric ribozyme-based gene control elements. (a) Each RNA switch will exhibit a dynamic range for ligand binding (DR_L) and for gene expression (DR_GE). The DR_GE will vary widely among synthetic or natural riboswitches. The DR_L should exhibit an 88-fold change in ligand concentration required to range from 10% to 90% gene expression for a riboswitch that carries a single aptamer domain, unless other factors are in play (e.g. non-linear cellular uptake of ligand; RNA folding problems). Dashed lines indicate the effects of imperfect gene control function, observed when the riboswitch RNA misfolds or otherwise fails to perfectly activate or repress gene expression even when folded correctly. (b) Comparison of theoretical DR_GE ranges reveals that there will be a biologically relevant DR_GE that natural or synthetic riboswitches need to encompass for their action to exhibit meaningful gene control. It is possible that synthetic riboswitches that exhibit the best DR_GE (large) might be less useful than a construct that has a far poorer DR_GE (small) that overlaps the biologically relevant range. (c) Sensitivity of ligand binding is critical for synthetic riboswitch utility. Less sensitive constructs might exhibit a DR_L that is outside the range of ligand concentrations that can be attained in cells. (d) Specificity of ligand recognition is critical for synthetic riboswitch utility. If the aptamer has a DRL that is orders of magnitude better that that for a close analog (DR_A) then gene control should be selectively triggered by the desired ligand if the analog is present in similar concentrations. However, if the analog naturally is present at concentrations that are orders of magnitude higher in concentration than the ligand, inappropriate regulation of gene expression may result.

Figure 4

Allosteric ribozymes as catalytic platforms for synthetic riboswitches. (a) Sequence and secondary structure model of an allosteric group I intron (Th2P6). Uppercase and lowercase letters identify intron and exon sequences, respectively. (b) A sequence encompassing only the minimal catalytic core of a hammerhead ribozyme with stems I through III identified. (c) Sequence and secondary structural model of the full-length Schistosoma mansoni hammerhead ribozyme. A construct based on this ribozyme is known to function in vivo. (d) Sequence and secondary structural model of a high-speed allosteric hammerhead ribozyme derived from the parental ribozyme in c. R1 through R3 (shaded nucleotides) were derived by in vitro selection from random-sequence domains. Theophylline binding is expected to permit the RNA to form tertiary contacts between the accessory domains in stems I and II and thereby exhibit high ribozyme activity.