Cell-free transcription-translation system: a dual read-out assay to characterize riboswitch function

Abstract

Cell-free protein synthesis assays have become a valuable tool to understand transcriptional and translational processes. Here, we established a fluorescence-based coupled in vitro transcription-translation assay as a read-out system to simultaneously quantify mRNA and protein levels. We utilized the well-established quantification of the expression of shifted green fluorescent protein (sGFP) as a read-out of protein levels. In addition, we determined mRNA quantities using a fluorogenic Mango-(IV) RNA aptamer that becomes fluorescent upon binding to the fluorophore thiazole orange (TO). We utilized a Mango-(IV) RNA aptamer system comprising four subsequent Mango-(IV) RNA aptamer elements with improved sensitivity by building Mango arrays. The design of this reporter assay resulted in a sensitive read-out with a high signal-to-noise ratio, allowing us to monitor transcription and translation time courses in cell-free assays with continuous monitoring of fluorescence changes as well as snapshots of the reaction. Furthermore, we applied this dual read-out assay to investigate the function of thiamine-sensing riboswitches thiM and thiC from Escherichia coli and the adenine-sensing riboswitch ASW from Vibrio vulnificus and pbuE from Bacillus subtilis, which represent transcriptional and translational on- and off-riboswitches, respectively. This approach enabled a microplate-based application, a valuable addition to the toolbox for high-throughput screening of riboswitch function.

Graphical Abstract

Figure 1.

Detection of mRNA and protein levels in cell-free transcription-translation systems using Mango-based fluorescence detection of mRNA and sGFP monitoring. (A) DNA template containing T7 regulatory elements, RBS, the sequence of sGFP with protein-coding start/stop sites and the Mango-(IV) RNA aptamer sequence as an array of one or four repeats and T7 terminator sequence. (B) Mango-(IV) RNA aptamer construct with the binding of the ligand TO3-acetate. (C) Excitation and emission spectra of sGFP and Mango-(IV) RNA aptamer fluorescent complex in the presence of TO3-acetate. (D) Mango-(IV) RNA aptamer sequence (M-IV) with TO3-acetate as ligand. For clarity, the G-quadruplex structure has been simplified. Mango-(IV) RNA aptamer array (M-IVx4) with mutated stem base pairs and a 5 nt linker sequence to prevent misfolding of individual aptamers. (E) Comparison of sGFP-M-IV and sGFP-M-IVx4 constructs at protein and mRNA levels. (F) Fluorescence binding curves for sGFP constructs with Mango-(IV) RNA aptamer monomer (M-IV) and Mango-(IV) RNA aptamer array (M-IVx4) were determined at a constant TO3-acetate ligand concentration (0.5 μM). Samples were measured in triplicate.

Figure 2.

TPP-sensing riboswitches thiM and thiC regulate transcription and translation in the context of TPP. (A) Schematic overview of the thiM riboswitch from E. coli representing the TPP-unbound and TPP-bound states. In the presence of TPP, the Shine-Dalgarno sequence (green) and AUG start-codon (red) are sequestered and not accessible to the ribosome. (B) Schematic overview of the thiC construct from E. coli representing the TPP-unbound and TPP-bound states. In the presence of TPP, translation initiation is inhibited by sequestration of the SD-sequence (green) and the AUG start-codon (red). (C) Time-dependent changes in mRNA levels monitored in a coupled transcription-translation assay of thiM (solid line) and thiC (dashed line) riboswitches with 0 and 100 μM TPP concentrations. Data were corrected with a negative control (without DNA) in the presence of 0.5 μM TO3-acetate. The samples were measured in triplicate, with a gain of 110 in the presence of 0.5 μM TO3-acetate. (D) mRNA monitoring of thiM and thiC in a coupled transcription-translation assay in the presence of 0 and 100 μM TPP. Data were corrected with a negative control (without DNA) in presence of 0.5 μM TO3-acetate. Results are shown for time points 0, 60 and 120 min and were measured in triplicate with a gain of 110 in the presence of 0.5 μM TO3-acetate. (E) sGFP monitoring of thiM and thiC in a coupled transcription-translation assay in the presence of 0 and 50 μM TPP. Data were corrected with a negative control (without DNA) in the presence of 0.5 μM TO3-acetate. Results are shown for time points 0, 60 and 120 min and were measured in triplicate with a gain of 70 in the presence of 0.5 μM TO3-acetate.

Figure 3.

Characterization of adenine-sensing riboswitch ASW in a coupled transcription-translation assay in the context of adenine. (A) Schematic overview of the three-state conformational equilibrium of the add ASW constructs from V. vulnificus. The mutated nucleotides of apoA and apoB are highlighted in blue, the SD-sequence in green, and the AUG start-codon in red. (B) Influence of adenine on the mRNA and protein levels of the control plasmid sGFP, containing a strong RBS. The coupled transcription-translation reactions were performed at 30°C and measured in triplicate with a gain of 50 for sGFP and 80 for mRNA. (C) sGFP and mRNA monitoring of ASW in a coupled transcription-translation assay in the presence and absence of adenine (1 mM) and TO3-acetate (0.5 μM). Reactions were performed at 30°C and measured in triplicate with a gain of 80 for sGFP and 110 for mRNA. (D) sGFP and mRNA monitoring of ASW, apoA and apoB in a coupled transcription-translation assay in the presence of 1 mM adenine and 0.5 μM TO3-acetate. Data were corrected with a negative control (without DNA) in the presence of 0.5 μM TO3-acetate. Reactions were performed at 30°C and measured in triplicate with a gain of 70 for sGFP and 110 for mRNA.