Engineering modular 'ON' RNA switches using biological components

Abstract

Riboswitches are cis-acting regulatory elements broadly distributed in bacterial mRNAs that control a wide range of critical metabolic activities. Expression is governed by two distinct domains within the mRNA leader: a sensory 'aptamer domain' and a regulatory 'expression platform'. Riboswitches have also received considerable attention as important tools in synthetic biology because of their conceptually simple structure and the ability to obtain aptamers that bind almost any conceivable small molecule using in vitro selection (referred to as SELEX). In the design of artificial riboswitches, a significant hurdle has been to couple the two domains enabling their efficient communication. We previously demonstrated that biological transcriptional 'OFF' expression platforms are easily coupled to diverse aptamers, both biological and SELEX-derived, using simple design rules. Here, we present two modular transcriptional 'ON' riboswitch expression platforms that are also capable of hosting foreign aptamers. We demonstrate that these biological parts can be used to facilely generate artificial chimeric riboswitches capable of robustly regulating transcription both in vitro and in vivo. We expect that these modular expression platforms will be of great utility for various synthetic biological applications that use RNA-based biosensors.

Figure 1.

Reengineering the B. subtilis pbuE adenine riboswitch. (a) Sequence and secondary structure of the wild-type pbuE riboswitch in the ‘OFF’ and ‘ON’ states. The red box denotes the nucleotides comprising the ligand-binding pocket, and the yellow box denotes sequence predicted to adopt adenine-dependent alternative secondary structures. Numbering is consistent with the experimental determination of the transcription start site of this riboswitch (48). (b) In vitro assay of effector-dependent transcriptional anti-termination using 2AP for the native B. subtilis pbuE riboswitch transcriptional unit (‘native’), the wild-type pbuE riboswitch under control of the T7A1 promoter (‘wild type’) and the pbuE(Δ1–11) mutant (‘Δ11’). A representative denaturing gel showing ³²P-labeled transcription products of the Δ11 mutant as a function of 2AP concentration is shown in the inset; ‘RT’ denotes the anti-terminated read through transcription product, and ‘T’ denotes the terminated product. Data plotted are the average of three independent measurements and the variation represented by the error bars. (c) Sequence and secondary structure of the pbuE ‘decoupled’ riboswitch capable of accommodating different aptamers. The cyan box denotes the insertion sequence that prevents the secondary structural switch from invading the aptamer domain. (d) In vitro transcription assay of the decoupled pbuE/pbuE* riboswitch along with two other aptamer chimeras. The inset demonstrates that the yitJ/pbuE* riboswitch regulates a transcriptional variation of the RNA and that a mutation (U78A) in the yitJ aptamer prevents SAM binding by the aptamer.

Figure 2.

Optimizing performance of the pbuE* expression platform. (a) Secondary structure of the core switching region of the pbuE* expression platform with sites of mutations introduced. (b) Graphical representation of quantified data from in vitro transcription assays of the C31G mutation for yitJ and xpt(C74U) aptamers fused to pbuE* expression platform. (c) Quantified data from in vitro transcription as a function of SAM concentration of yitJ/pbuE* chimeras incorporating deletions of 0, 1, 2 or 3 uridines from the poly-uridine tract. (d) Quantified percentage read through transcription of xpt(C74U)/pbuE* chimeras as a function of 2AP concentration. The insets show two representative denature gels of transcription of chimeras with the wild-type expression platform (bottom) and the deletion of three uridines (top). The data plotted is the average of three independent experiments, and the error bars represent the uncertainty in the measurement.

Figure 3.

Activity of chimeric riboswitches with the pbuE* expression platform module. (a) In vitro transcription of various chimeric riboswitches as a function of their cognate effector ligand. Each chimera incorporates the pbuE* module with the ΔU126 (7U) mutation that affects termination efficiency. The data plotted are the averages of three independent experiments, and the standard deviation of these measurements are represented by the error bars. (b) Titrations of the same set of chimeras but with the ΔU126–127 (6U) mutation in the pbuE* expression platform. Fitted values for T₅₀ and %RT_min are given in Table 1.

Figure 4.

Reengineering the SAH-dependent metH riboswitch. (a) Sequence and secondary structure of the ‘OFF’ and ‘ON’ states of sahH/metH riboswitch. Poly-uridine tract mutations made in this study are denoted in the structure on the left; the yellow box denotes sequence overlap between the aptamer domain and expression platform. The arrowheads denote the break points for splicing in foreign aptamers (see panel c). (b) Activity of the native D. aromatica metH riboswitch (metH, red), the sahH/metH chimeric riboswitch (blue) and the sahH/metH* chimera incorporating the G128U,C129U mutation (black). (c) Sequence and secondary structure of the chimeric xpt/metH* riboswitch in the ‘OFF’ and ‘ON’ states. The dashed line denotes the boundary between the two domains that is used for making all metH chimeras. The C74U mutation that changes the aptamers selectivity from guanine to adenine and 2AP is denoted along with the ‘CTS’ mutation used for in vivo analysis of this riboswitch (Figure 6) is denoted. (d) Quantified activity assay of the xpt/metH* riboswitch with representative gel of transcription products shown in the inset.

Figure 5.

Optimization of metH expression platform performance. (a) Mutations introduced into the metH* platform to repress read through transcription at low effector concentration. Wild-type sequence is highlighted in cyan and mutations [P1(−1), P1(−2) and P1(−3)] in orange. The yellow box denotes the switching sequence. (b) In vitro transcription of xpt/metH* and P1 mutants. The percentage read through product (%RT) in the absence and presence of 1 µM guanine are shown along with the DR. (c) Full titrations of four chimeras of the metH^‡ expression platform [xpt, xpt(C74U), tetracycline, sahH] titrated with their cognate effector ligands. The data points represent the average of three independent titrations.

Figure 6.

In vivo characterization of pbuE* and metH^‡ chimeric riboswitches. (a) Quantification of induction of GFPuv expression as a function of 2AP concentration in a defined medium. The induction factor represents the fold increase in normalized GFP expression of the reporter under control of a riboswitch over the same riboswitch under no ligand conditions. Data was fit to a two-state model and plotted as a function of 2AP to yield the EC₅₀. The U89A mutation (blue diamonds) is a mutant that is defective in 2AP binding as a negative control. (b) Induction of GFP expression by the pbuE/pbuE* chimera (‘8U’, blue) and uridine-rich tract mutations (5U, 6U and 7U). (c) The same series of experiments performed with xpt(C74U)/pbuE* chimeras. The pink triangles represent a U51C mutant of xpt(C74U)/pbuE* that is deficient in ligand binding. (d) Activity of the xpt(C74U)/metH chimera (black) and the ‘CTS’ mutant (blue). Nonbinding mutants are denoted in green and pink as negative controls. The data presented is the average of at least three independent experiments and the standard deviation of the average value is represented as the error bars.