Direct evaluation of tRNA aminoacylation status by the T-box riboswitch using tRNA-mRNA stacking and steric readout

Abstract

T-boxes are gene-regulatory mRNA elements with which Gram-positive bacteria sense amino acid availability. T-boxes have two functional domains. Stem I recognizes the overall shape and anticodon of tRNA, while a 3' domain evaluates its aminoacylation status, overcoming an otherwise stable transcriptional terminator if the bound tRNA is uncharged. Although T-boxes are believed to evaluate tRNA charge status without using any proteins, this has not been demonstrated experimentally because of the instability of aminoacyl-tRNA. Using a simple method to prepare homogeneous aminoacyl-tRNA, we show that the Bacillus subtilis glyQS T-box functions independently of any tRNA-binding protein. Comparison of aminoacyl-tRNA analogs demonstrates that the T-box detects the molecular volume of tRNA 3'-substituents. Calorimetry and fluorescence lifetime analysis of labeled RNAs shows that the tRNA acceptor end coaxially stacks on a helix in the T-box 3' domain. This intimate intermolecular association, selective for uncharged tRNA, stabilizes the antiterminator conformation of the T-box.

Figure 1. An Efficient Method to Prepare Homogeneous aa-tRNAs

(A) Aminoacylation of tRNA and protection of aa-tRNA. The overall yield (on a tRNA basis) for the two steps is 50–60%. (B) Protection of the α-amino group of aa-tRNA alters its gel mobility. (C) C18 RP-HPLC chromatogram of the pentenoylation reaction mixture. (D) Acid-PAGE analysis of fractions from (C). (E) Mild deprotection of protected aa-tRNA regenerates aa-tRNA. (F) Acid-PAGE analysis of the purified, deprotected aa-tRNA. See also Figure S1.

Figure 2. Mechanisms of tRNA 3′ End Recognition by a T-box Riboswitch

(A) DNA template used for the in vitro antitermination assay. Transcription elongation complexes (ECs) were synchronized at position 16 by withholding CTP. Purified EC16 was incubated with 3 μM tRNA, before restarting by adding 100 μM of all NTPs. (B) Representative Urea-PAGE analysis showing tRNA aminoacylation-dependent transcriptional discrimination by the T-box. (C) Top, a panel of tRNA variants (Figure S2). Bottom, fraction of transcription readthrough induced by nine tRNA variants. Error bars represent s.d. from three independent experimental replicates. (D) Top, a panel of tRNA variants carrying a range of 3′ substituents. Bottom, plot of fraction of transcription readthrough against the van der Waals volume of each tRNA 3′ substituent. Pearson’s R² was computed omitting the protected-aa-tRNA. Error bars represent s.d. from three independent experimental replicates.

Figure 3. 2AP Fluorescence Lifetimes Reveal the Structural Context of the tRNA 3′ End Bound to the T-box Antiterminator

(A) Normalized traces of 2AP fluorescence decay and best fits for RNAs in (B). IRF: instrument response function. (B) Secondary structures of RNAs analyzed in (A). Scissors indicate nick position in the split T-box reconstituted with T-box¹⁷⁰ and T-box^171–182. (C) Average 2AP fluorescence lifetimes of the RNAs shown in (B), as colored in (A). See also Figure S3. Error bars represent s.d. from three independent experimental replicates.

Figure 4. PyC Fluorescence Lifetime and Calorimetric Analyses Suggest Coaxial Stacking between the tRNA 3′ end and Antiterminator Helix A1

(A) Normalized traces of PyC fluorescence decay and best fits for RNAs in (B). (B) Secondary structures of RNAs analyzed in (A). (C) Average PyC lifetimes of the RNAs shown in (B), as colored in (A). Error bars represent s.d. from three independent experimental replicates. See also Figure S3. (D) Secondary structure schematic of a truncated tRNA-T-box complex without helix A2. (E) Two-step ITC analysis of the energetic contribution from the proposed stacking between tRNA 3′ end tA76 and T-box C178. The upper panel explains the sequential ITC experiment, and the lower panel shows the K_d (blue) and enthalpy changes (orange) from the ITC titrations (Figure S4). Error bars represent s.d. from three independent replicates. (*, p=0.052; ***, p=0.0011; two-tailed t-test). See also Figure S4. (F) Model of a full-length T-box in complex with uncharged tRNA based on this work, phylogenetic analysis, and the co-crystal structure of a T-box Stem I bound to its cognate tRNA (Zhang and Ferré-D’Amaré, 2013). tRNA is in green, T-box in blue, 7-nt antiterminator bulge in purple, aminoacylation site in magenta. Stacked cylinders denote coaxially stacked RNA helices.