De novo design of a synthetic riboswitch that regulates transcription termination

Abstract

Riboswitches are regulatory RNA elements typically located in the 5'-untranslated region of certain mRNAs and control gene expression at the level of transcription or translation. These elements consist of a sensor and an adjacent actuator domain. The sensor usually is an aptamer that specifically interacts with a ligand. The actuator contains an intrinsic terminator or a ribosomal binding site for transcriptional or translational regulation, respectively. Ligand binding leads to structural rearrangements of the riboswitch and to presentation or masking of these regulatory elements. Based on this modular organization, riboswitches are an ideal target for constructing synthetic regulatory systems for gene expression. Although riboswitches for translational control have been designed successfully, attempts to construct synthetic elements regulating transcription have failed so far. Here, we present an in silico pipeline for the rational design of synthetic riboswitches that regulate gene expression at the transcriptional level. Using the well-characterized theophylline aptamer as sensor, we designed the actuator part as RNA sequences that can fold into functional intrinsic terminator structures. In the biochemical characterization, several of the designed constructs show ligand-dependent control of gene expression in Escherichia coli, demonstrating that it is possible to engineer riboswitches not only for translational but also for transcriptional regulation.

Figure 1.

Riboswitch constructs. (A) Design strategy for theophylline-dependent riboswitches controlling transcription. The sequence of the TCT8-4 theophylline aptamer (red) was fused to a short spacer region (cyan) followed by a sequence complementary to the 3′-part of the aptamer (blue) and a U stretch (black). The Shine–Dalgarno sequence (black box) and the open reading frame of the reporter gene bgaB are located immediately downstream of this construct. On theophylline binding, terminator structure formation should be inhibited and transcription should proceed, resulting in expression of the reporter gene. (B) The final riboswitch constructs are transcribed from the arabinose promoter of plasmid pBAD. The transcription start point is indicated by the angled arrow. The Shine–Dalgarno sequence (AGGA) of the reporter gene for β-galactosidase is located immediately downstream of the riboswitch insert. The start codon of the reporter gene is underlined.

Figure 2.

Riboswitch constructs and activity tests. (A) The theophylline aptamer represents the sensor (red), followed by a variable spacer sequence (cyan) and the 3′-part of the terminator (blue), forming a hairpin structure with a subsequence of the sensor domain. The terminator element is completed by a stretch of eight U residues (black). Below each construct, dot bracket notations of the secondary structures with terminator or antiterminator formation are indicated. The overlapping situation of sensor (theophylline aptamer, red box) and actuator element (terminator element, grey box) is required for a mutual exclusion of the two alternative structures. For each construct, calculated energy values of complete riboswitch elements (RS) and isolated terminators (T) are indicated. (B) Activity tests of the reporter enzyme β-galactosidase. Activities are indicated in Miller units (MU). All synthetic riboswitches were tested in the absence (black bars) and presence (gray bars) of 2 mM theophylline. As a control, the structurally related caffeine was offered (white bars). (C) Activity test of RS10 under the control of the Prrn promoter and GFP as reporter gene. Relative fluorescence signals are indicated as a function of theophylline concentration. The riboswitch shows a similar regulation profile as in (B), indicating the general functionality of the construct.

Figure 3.

Optimized RS10 and RS3 variants and control constructs. (A) In RS10loop, the tetraloop sequence of the terminator hairpin was adjusted to the frequently found sequence GAAA, including a closing G-C base pair. In RS10shift, a spacer sequence of 19 nt was inserted between the U₈ stretch of the terminator and the Shine–Dalgarno sequence (black box). The same 19 nt spacer was inserted in RS3shift at the identical position. In control bgaB, the upstream untranslated region of the reporter gene did not carry any riboswitch or terminator inserts. Furthermore, the U₈ stretch alone was placed immediately upstream of the ribosomal binding site, and the 19 nt spacer sequence region was inserted into the original bgaB control construct, leading to bgaBU8 and bgaBshift, respectively. (B) β-galactosidase activity test of the constructs described in (A). Activities are indicated in Miller units (MU). The tetraloop adaptation (RS10loop) in the terminator element led to a reduction of the background activity but lowered also the expression of the reporter gene. Increasing the distance between the riboswitch U₈ stretch and the ribosomal binding site of the reporter gene in RS10shift and RS3shift increased the ON/OFF rate substantially, compared with the original constructs (for comparison, the regulation profiles of RS3 and RS10 shown in Figure 2B are indicated in this panel). In the control elements, the U₈ stretch immediately upstream of the ribosomal binding site led to a rather high gene expression (bgaBU₈), whereas increasing the distance between these sequences reduced the β-galactosidase activity (bgaBshift).

Figure 4.

Functional analysis of RS10 and synthetic terminator elements. (A) The isolated terminators of the tested riboswitch constructs were cloned upstream of the reporter gene. In a control construct, the 3′-complementary sequence of terminator 10 and the U₈ stretch was deleted, leading to RS10ΔT. As shown in Figure 3, construct bgaBU₈ represents the positive control. (B) β-galactosidase activity of terminator constructs. Although the positive control bgaBU₈ (activation data from Figure 3B are presented) and the terminator deletion RS10ΔT show a strong enzyme activity, all terminators reduce the β-galactosidase activity at an efficiency comparable with that of the natural terminator from the pyrBI operon in E. coli (pyrBI). The terminator elements of the functional riboswitches 1, 3 and 10 show the strongest activity reduction. (C) Northern blot analysis of RS10. Left panel: Although the terminator-alone construct T10 showed no transcription of the reporter gene, RS10 allowed bgaB gene transcription exclusively in the presence (+) of theophylline. C, control expression of bgaB using pBAD2-bgaB without riboswitch inserts. 23S rRNA was used as an internal standard for normalizing the presented lanes. Right panel: Quantitation of bgaB transcription in RS10 construct with and without theophylline.

Figure 5.

A possible correlation between terminator stability and riboswitch function. Among all tested terminators, T8 has the lowest free energy value and forms a stable structure that obviously cannot be disrupted on ligand binding. As a consequence, RS8 is locked in an OFF state. In contrast, T2 and T4 form a much weaker hairpin, shifting the equilibrium towards the aptamer structure that interferes with terminator formation. The functional riboswitches RS1, RS3 and RS10 show intermediate stabilities of their terminator elements that allow the desired ligand-dependent rearrangement of the constructs, switching between ON and OFF states. The stabilized terminator hairpin of RS10loop is located between the terminators of RS8 and the functional RS3. Although this stabilized hairpin reduces the background transcription rate considerably, it obviously impedes the structural rearrangement induced by ligand binding and allows only a moderate level of gene expression.