Factors that influence T box riboswitch efficacy and tRNA affinity

Abstract

The T box riboswitch is an intriguing potential target for antibacterial drug discovery. Found primarily in Gram-positive bacteria, the riboswitch regulates gene expression by selectively responding to uncharged tRNA to control transcription readthrough. Polyamines and molecular crowding are known to specifically affect RNA function, but their effect on T box riboswitch efficacy and tRNA affinity have not been fully characterized. A fluorescence-monitored in vitro transcription assay was developed to readily quantify these molecular interactions and to provide a moderate-throughput functional assay for a comprehensive drug discovery screening cascade. The polyamine spermidine specifically enhanced T box riboswitch readthrough efficacy with an EC50 = 0.58 mM independent of tRNA binding. Molecular crowding, simulated by the addition of polyethylene glycol, had no effect on tRNA affinity for the riboswitch, but did reduce the efficacy of tRNA-induced readthrough. These results indicate that the T box riboswitch tRNA affinity and readthrough efficacy are intricately modulated by environmental factors.

Keywords: Drug discovery; Molecular crowding; RNA; Spermidine; T box riboswitch.

Figure 1. Schematic of glyQS T box riboswitch and fluorescence-monitored transcription readthrough

a) In the absence of tRNA^Gly transcription terminates prior to the RNA sequence targeted by the fluorescent probe RT_prb. The hairpin form of RT_prb has minimal background fluorescence due to Cy5 fluorescence being quenched by Dabcyl. b) Transcription readthrough induced by tRNA^Gly produces the full-length transcript, including the target sequence of RT_prb. RT_prb binds the target sequence, unfolding the hairpin structure such that Dabcyl no longer quenches Cy5 and fluorescence increases.

Figure 2. Multi-round assay for tRNA^Gly-specific induction of glyQS transcription readthrough

a) Comparison of Readthrough_max of cognate tRNA^Gly (wt, acceptor end 5′-UCCA-3′) with tRNA^Gly(U73A), tRNA^Tyr(A73U) and tRNA^Tyr(wt, acceptor end 5′-ACCA-3′). For all reactions with tRNA, [tRNA] = 100 nM. b) Dose-response curve of tRNA^Gly readthrough induction.

Figure 3. Spermidine effect on efficacy of tRNA^Gly-induced glyQS readthrough

a) Structure of spermidine. b) Spermidine dose-response effect on glyQS transcription readthrough. In the absence of tRNA^Gly (open circle), spermidine EC₅₀ = 0.49 mM (95% CI = 0.41-0.59 mM, R² = 0.96). In the presence of tRNA^Gly(filled circle), spermidine EC₅₀ = 0.58 mM (95% CI = 0.49-0.68 mM, R² = 0.98). The Transcription Control data (triangle) reflect the overall transcription reaction since the Transcription Control DNA template lacks the T box riboswitch. For the Transcription control, the spermidine EC₅₀ = 0.82 mM (95% CI = 0.74-0.91 mM, R² = 0.95). The data points at [spermidine] = 10 mM were excluded from EC₅₀ calculations as discussed in Results.

Figure 4. Molecular crowding effect on tRNA^Gly-induced glyQS readthrough

a) Transcription Control (striped bars), glyQS readthrough in the presence of 100 nM tRNA^Gly (shaded bars), and glyQS readthrough in the absence of tRNA^Gly (basal level transcription, unfilled bars) were performed in the presence or absence of 5% (wt/v) PEG-8000. The Transcription Control data reflect the overall transcription reaction since the Transcription Control DNA template lacks the T box riboswitch. b) Effect of molecular crowding on the affinity (EC₅₀) of tRNA^Gly to induce glyQS readthrough. For tRNA^Gly EC₅₀ = 30 nM in the absence of PEG (open circle, 95% CI = 22-39 nM, R² = 0.97) and EC₅₀ = 26 nM in the presence of 5% PEG (filled circle, 95% CI = 13-50 nM, R² = 0.84).

Figure 5. Effect of 1, 4-disubstutited 1, 2, 3-triazole on glyQS transcription readthrough

a) Dose-response of compound 1 on glyQS transcription readthrough in the presence of 15 nM tRNA^Gly. Apparent IC₅₀ = 8 μM (95% CI = 4-15 μM, R² = 0.86). Inset: Structure of triazole 1. b) Readthrough_max without (open bars) and with 1 (50 μM, shaded bars) for glyQS basal level transcription, glyQS with tRNA^Gly (15 nM), and, the Transcription Control reaction. For each reaction condition, the percent of ligand-induced inhibition is indicated. The Transcription Control data reflect the effect of 1 on the overall transcription reaction since the Transcription Control DNA template lacks the T box riboswitch. Also shown is the effect of 1 on RT_prb fluorescence in the presence of Target Sequence RNA (20 nM).