Journal: eLife

Article Title: Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

doi: 10.7554/eLife.39518

Figure Lengend Snippet: ( A ) FRET labeling scheme for the T-box and tRNA. Cy3 (green star) and Cy5 (red star) fluorophores are attached at the 3’ of the T-box (blue) and the 5’ of the tRNA (black), respectively. glyQS T-box riboswitch molecules are anchored on slides through a biotinylated DNA probe (purple) hybridized to a 5’ extension sequence on the T-box. ( B ) smFRET vs. time trajectories of T-box 182 -Cy3(3’) with tRNA Gly -Cy5, T-box 182 -Cy3(3’) with tRNA ΔNCCA -Cy5 and T-box 149 -Cy3(3’) with tRNA Gly -Cy5. Cy3 and Cy5 fluorescence intensity traces (upper panel), and their corresponding smFRET traces calculated as I Cy5 / (I Cy3+ I Cy5 ) (lower panel). ( C ) One-dimensional FRET histograms. FRET peaks are fit with a Gaussian distribution (black curve) and the peak centers are shown in red. ‘N’ denotes the total number of traces in each histogram from three independent experiments. ( D ) Transition density plot (TDP). Contours are plotted from white (less than 15% of the maximum population) to red (more than 85% of the maximum population). TDPs are generated from all smFRET traces from three independent experiments. ( E ) Representative smFRET trajectories showing real-time binding of tRNA Gly -Cy5 to T-box 182 -Cy3(3’) in a steady-state measurement. Traces showing transitions from the unbound state (0 FRET) to fully bound state (0.7 FRET) through the partially bound state (0.4 FRET) (left) and unbound state directly to fully bound state (right). ( F ) Surface contour plot of time-evolved FRET histogram of T-box 182 -Cy3(3’) with tRNA Gly -Cy5 (top) and tRNA ΔNCCA -Cy5 (bottom). Contours are plotted from blue (less than 5% of the maximum population) to red (more than 75% of the maximum population). ‘N’ denotes the total number of traces in each histogram from three independent experiments, which are a subset of traces showing real-time binding events in the steady-state measurements. Total numbers of traces in steady-state measurements are indicated in (C). Traces that reach the 0.7 FRET state (cutoff >0.55) are included in the plot for tRNA Gly -Cy5 to reveal better the transition from the 0.4 to the 0.7 FRET state. Time-evolved FRET histograms of all traces are shown in for comparison. 10.7554/eLife.39518.014 Figure 2—source data 1. Cy3 and Cy5 intensity traces from three repeats for T-box 182 -Cy3(3’) and tRNA Gly -Cy5. Each two columns show intensities of the Cy3/Cy5 pair from the same molecule. 10.7554/eLife.39518.015 Figure 2—source data 2. Cy3 and Cy5 intensity traces from three repeats for T-box 182 -Cy3(3’) and tRNA ΔNCCA -Cy5. 10.7554/eLife.39518.016 Figure 2—source data 3. Cy3 and Cy5 intensity traces from three repeats for T-box 149 -Cy3(3’) and tRNA Gly -Cy5.

Article Snippet: The RNA was incubated with Cy3 hydrazide (Lumiprobe) dye for 45 min with a final RNA/dye ratio of ~1:200.

Techniques: Labeling, Sequencing, Fluorescence, Generated, Binding Assay, Comparison

Journal: eLife

Article Title: Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

doi: 10.7554/eLife.39518

Figure Lengend Snippet: Images were created by maximum intensity projection of Cy5 emission of the time-lapse images and therefore report on the binding events of tRNA-Cy5 to the surface tethered T-box. A DNA oligo labeled with Cy3 is used as a negative control for non-specific binding or signal. Loading tRNA Tyr -Cy5 to pre-immobilized T-box-Cy3(3’) only generates background level of Cy5 signals in the maximum intensity projection similar to the negative control, and these nonspecific Cy5 signals do not generate any smFRET traces.

Article Snippet: The RNA was incubated with Cy3 hydrazide (Lumiprobe) dye for 45 min with a final RNA/dye ratio of ~1:200.

Techniques: Binding Assay, Labeling, Negative Control

Journal: eLife

Article Title: Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

doi: 10.7554/eLife.39518

Figure Lengend Snippet: ( A ) Dwell time of i) 0.7 FRET state to 0.4 FRET state, ii) 0.4 FRET state to 0.7 FRET state, iii) 0.7 FRET state to 0 FRET state, and iv) 0.7 FRET state to other FRET states of T-box 182 -Cy3(3’) with tRNA Gly -Cy5. Histograms of i), ii), and iii) are fit with a single-exponential decay function (black curve) and iv) is fit with a double exponential decay function to generate the population-weighted average lifetime of the 0.7 FRET state (τ 0.7 ), as molecules can transit from 0.7 FRET state to both 0.4 FRET state occasionally, and 0 FRET state upon fluorophore photobleaching. ( B ) Dwell time of i) 0 FRET to 0.4 FRET state and ii) 0.4 FRET state to 0 FRET of T-box 182 -Cy3(3’) with tRNA ΔNCCA -Cy5. Histograms are fit with a single-exponential decay function (black curve). ( C ) Dwell time of i) 0 FRET state to 0.4 FRET state and ii) 0.4 FRET state to 0 FRET state of T-box 149 -Cy3(3’) with tRNA Gly -Cy5. Histograms are fit with a single-exponential decay function (black curve). Mean ±standard deviation (S.D.) are calculated from three independent measurements.

Article Snippet: The RNA was incubated with Cy3 hydrazide (Lumiprobe) dye for 45 min with a final RNA/dye ratio of ~1:200.

Techniques: Standard Deviation

Journal: eLife

Article Title: Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

doi: 10.7554/eLife.39518

Figure Lengend Snippet: ( A ) Ribbon diagram of a complex of the B. subtilis glyQS T-box riboswitch and uncharged tRNA Gly . Distances from the 5’ end of the tRNA to the 3’ end of the anti-terminator (T-box 182 ) and to the 3’ end of Stem III (T-box 149 ) are marked. ( B ) One dimensional FRET histograms of intra-T-box pair of T-box 182 (black) and T-box 149 (red) with Cy3 attached directly to the 3’ end of the T-box and Cy5 attached to the oligo hybridized to the 5’ extension of the T-box. Only the first 50 data points of the FRET trajectories are used to plot the histogram to eliminate the zero FRET resulting from Cy5 photobleaching. ‘N’ denotes the total number of traces in each histogram from two independent experiments. 10.7554/eLife.39518.010 Figure 2—figure supplement 3—source data 1. Cy3 and Cy5 intensity traces from two repeats for intra-T-box 182 FRET in the absence of tRNA. 10.7554/eLife.39518.011 Figure 2—figure supplement 3—source data 2. Cy3 and Cy5 intensity traces from two repeats for intra-T-box 149 FRET in the absence of tRNA.

Article Snippet: The RNA was incubated with Cy3 hydrazide (Lumiprobe) dye for 45 min with a final RNA/dye ratio of ~1:200.

Techniques:

Journal: eLife

Article Title: Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

doi: 10.7554/eLife.39518

Figure Lengend Snippet: ( A ) smFRET trajectories of T-box 182 -Cy3(3’) and tRNA Gly -Cy5. Black arrows represent the time point when tRNA Gly -Cy5 is flowed through the imaging area. Dwell time of unbound state (T off ) was calculated as the time between starting point and first FRET transition. ( B ) Surface contour plot of time-evolved FRET histogram of the flow experiment. ‘N’ denotes the total number of traces in each histogram from two independent experiments. ( C ) Histogram of τ off and its single-exponential decay fitting. Mean ±S.D. are calculated from two independent measurements. ( D ) Surface contour plot of time-evolved FRET histogram of all traces showing real-time binding events, including the traces that are unable to reach the 0.7 FRET state. The same data sets are used in . ‘N’ denotes the total number of traces in each histogram from three independent experiments, which are a subset of traces showing real-time binding event in the steady-state measurements. Total numbers of traces in the steady-state measurements are indicated in . Contours in ( B ) and ( D ) are plotted in the same way as in . 10.7554/eLife.39518.013 Figure 2—figure supplement 4—source data 1. Cy3 and Cy5 intensity traces from two repeats for T-box 182 -Cy3(3’) and tRNA Gly -Cy5 in the flow experiments.

Article Snippet: The RNA was incubated with Cy3 hydrazide (Lumiprobe) dye for 45 min with a final RNA/dye ratio of ~1:200.

Techniques: Imaging, Binding Assay

Journal: eLife

Article Title: Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

doi: 10.7554/eLife.39518

Figure Lengend Snippet: ( A ) Cy5 (red star) and Cy3 (green star) are attached at the 5’ extension of the T-box (blue) and the 3’ end of the T-box, respectively. ( B ) One dimensional FRET histograms of intra-T-box pair of T-box 182 alone (black) and with tRNA Gly (red). ‘N’ denotes the total number of traces in each histogram from all independent experiments. For ‘-tRNA’ case, two independent measurements were performed. For ‘+tRNA Gly ’ case, as no difference was detected, we only performed the measurement once. 10.7554/eLife.39518.019 Figure 3—figure supplement 1—source data 1. Cy3 and Cy5 intensity traces for intra-T-box 182 FRET in the presence of tRNA Gly .

Article Snippet: The RNA was incubated with Cy3 hydrazide (Lumiprobe) dye for 45 min with a final RNA/dye ratio of ~1:200.

Techniques:

Journal: eLife

Article Title: Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

doi: 10.7554/eLife.39518

Figure Lengend Snippet: ( A ) Intra-T-box FRET scheme. Cy3 (green star) and Cy5 (red star) are attached at the 5’ and 3’ extensions of T-box (blue), respectively. ( B ) One-dimensional FRET histograms of T-box 182 alone, with tRNA Phe , with tRNA ΔNCCA , and with tRNA Gly . ‘N’ denotes the total number of traces in each histogram from three independent experiments. 10.7554/eLife.39518.020 Figure 3—source data 1. Cy3 and Cy5 intensity traces from three repeats for intra T-box 182 FRET with 3’ extension without tRNA. 10.7554/eLife.39518.021 Figure 3—source data 2. Cy3 and Cy5 intensity traces from three repeats for intra T-box 182 FRET with 3’ extension in the presence of tRNA Phe . 10.7554/eLife.39518.022 Figure 3—source data 3. Cy3 and Cy5 intensity traces from three repeats for intra T-box 182 FRET with 3’ extension in the presence of tRNA ΔNCCA . 10.7554/eLife.39518.023 Figure 3—source data 4. Cy3 and Cy5 intensity traces from three repeats for intra T-box 182 FRET with 3’ extension in the presence of tRNA Gly .

Article Snippet: The RNA was incubated with Cy3 hydrazide (Lumiprobe) dye for 45 min with a final RNA/dye ratio of ~1:200.

Techniques:

Journal: eLife

Article Title: Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

doi: 10.7554/eLife.39518

Figure Lengend Snippet: ( A ) Cy3 (green star) and Cy5 (red star) are attached at the 5’ extension of the T-box (blue) and the 5’ of the tRNA (black), respectively. ( B ) smFRET trajectories of T-box-Cy3(5’) with tRNA ΔNCCA -Cy5 (left) and tRNA Gly -Cy5 (right). ‘N’ denotes the total number of traces in each histogram from three independent experiments. ( C ) One-dimensional FRET histograms of T-box-Cy3(5’) with tRNA ΔNCCA -Cy5 (left) and tRNA Gly -Cy5 (right). ( D ) TDP of T-box-Cy3(5’) with tRNA ΔNCCA -Cy5 (left) and tRNA Gly -Cy5 (right). Contours are plotted in the same way as in . TDPs are generated from all smFRET traces from three independent experiments. 10.7554/eLife.39518.026 Figure 4—source data 1. Cy3 and Cy5 intensity traces from three repeats for T-box 182 -Cy3(5’) with tRNA Gly -Cy5. 10.7554/eLife.39518.027 Figure 4—source data 2. Cy3 and Cy5 intensity traces from three repeats for T-box 182 -Cy3(5’) with tRNA ΔNCCA -Cy5.

Article Snippet: The RNA was incubated with Cy3 hydrazide (Lumiprobe) dye for 45 min with a final RNA/dye ratio of ~1:200.

Techniques: Generated

Journal: eLife

Article Title: Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

doi: 10.7554/eLife.39518

Figure Lengend Snippet: ( A ) Schematic diagram of three different mutations introduced to the T-box 182 backbone (T-box C56U , T-box SIII-Δ4bp and T-box ΔKT ). ( B ) Representative smFRET traces of T-box ΔKT -Cy3(3’) and tRNA Gly -Cy5. ( C ) FRET histograms of the T-box mutants with tRNA Gly -Cy5. ‘N’ denotes the total number of traces in each histogram from three independent experiments. ( D ) TDP of the T-box mutants with tRNA Gly -Cy5. TDPs are generated from all smFRET traces from three independent experiments and are plotted in the same way as in . (E) Table of kinetic parameters for tRNA Gly -Cy5 binding to different T-box constructs. k -1 , k 2 , and k -2 of T-box ΔKT -Cy3(3’) are apparent rate constants estimated to allow comparison as described in Materials and methods. All rate constants are reported as mean ± standard deviation (S.D.) from three or four independent experiments. 10.7554/eLife.39518.035 Figure 6—source data 1. Cy3 and Cy5 intensity traces from three repeats for T-box C56U -Cy3(3’) with tRNA Gly -Cy5. 10.7554/eLife.39518.036 Figure 6—source data 2. Cy3 and Cy5 intensity traces from four repeats for T-box SIII-Δ4bp -Cy3(3’) with tRNA Gly -Cy5. 10.7554/eLife.39518.037 Figure 6—source data 3. Cy3 and Cy5 intensity traces from three repeats for T-box ΔKT -Cy3(3’) with tRNA Gly -Cy5. 10.7554/eLife.39518.038 Figure 6—source data 4. Cy3 and Cy5 intensity traces from three repeats for T-box C56U -Cy3(3’) with tRNA ΔNCCA -Cy5. 10.7554/eLife.39518.039 Figure 6—source data 5. Cy3 and Cy5 intensity traces from three repeats for T-box SIII-Δ4bp -Cy3(3’) with tRNA ΔNCCA -Cy5. 10.7554/eLife.39518.040 Figure 6—source data 6. Cy3 and Cy5 intensity traces from three repeats for T-box ΔKT -Cy3(3’) with tRNA ΔNCCA -Cy5.

Article Snippet: The RNA was incubated with Cy3 hydrazide (Lumiprobe) dye for 45 min with a final RNA/dye ratio of ~1:200.

Techniques: Generated, Binding Assay, Construct, Comparison, Standard Deviation

Journal: eLife

Article Title: Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

doi: 10.7554/eLife.39518

Figure Lengend Snippet: ( A ) Surface contour plot of time-evolved FRET histogram of T-box C56U -Cy3(3’) with tRNA Gly -Cy5 with traces showing real-time binding. Contours are plotted in the same way as in . ‘N’ denotes the total number of traces in each histogram from three independent experiments, which are a subset of traces showing real-time binding event in the steady-state measurements. Total number of traces in the steady-state measurements is indicated in . (B) FRET histogram of T-box C56U -Cy3(3’) with tRNA ΔNCCA -Cy5. ‘N’ denotes the total number of traces three independent experiments. ( C ) TDP of T-box C56U -Cy3(3’) with tRNA ΔNCCA -Cy5. TDP is generated from all smFRET traces from three independent experiments and is plotted in the same way as in .

Article Snippet: The RNA was incubated with Cy3 hydrazide (Lumiprobe) dye for 45 min with a final RNA/dye ratio of ~1:200.

Techniques: Binding Assay, Generated

Journal: eLife

Article Title: Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

doi: 10.7554/eLife.39518

Figure Lengend Snippet: ( A ) Surface contour plot of time-evolved FRET histogram of T-box SIII-Δ4bp -Cy3(3’) with tRNA Gly -Cy5 with traces showing real-time binding. Contours are plotted in the same way as in . ‘N’ denotes the total number of traces in each histogram from three independent experiments, which are a subset of traces showing real-time binding event in the steady-state measurements. Total number of traces in the steady-state measurements is indicated in . (B) FRET histogram of T-box SIII-Δ4bp -Cy3(3’) with tRNA ΔNCCA -Cy5. ‘N’ denotes the total number of traces from four independent experiments. ( C ) TDP of T-box SIII-Δ4bp -Cy3(3’) with tRNA ΔNCCA -Cy5. TDP is generated from all smFRET traces from four independent experiments and is plotted in the same way as in .

Article Snippet: The RNA was incubated with Cy3 hydrazide (Lumiprobe) dye for 45 min with a final RNA/dye ratio of ~1:200.

Techniques: Binding Assay, Generated

Journal: eLife

Article Title: Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

doi: 10.7554/eLife.39518

Figure Lengend Snippet: ( A ) FRET histogram of T-box ΔKT -Cy3(3’) with tRNA ΔNCCA -Cy5. ‘N’ denotes the total number of traces from three independent experiments. ( B ) TDP of T-box ΔKT -Cy3(3’) with tRNA ΔNCCA -Cy5. TDP is generated from all smFRET traces from three independent experiments and is plotted in the same way as in . (C) Kinetic model of tRNA Gly binding to T-box ΔKT . Transition rate constants are reported as mean ±S.D from three independent measurements. Probabilities of different pathways for transitioning into and out of 0.7 FRET states are marked in red in parenthesis.

Article Snippet: The RNA was incubated with Cy3 hydrazide (Lumiprobe) dye for 45 min with a final RNA/dye ratio of ~1:200.

Techniques: Generated, Binding Assay

Journal: eLife

Article Title: Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

doi: 10.7554/eLife.39518

Figure Lengend Snippet:

Article Snippet: The RNA was incubated with Cy3 hydrazide (Lumiprobe) dye for 45 min with a final RNA/dye ratio of ~1:200.

Techniques: Recombinant, Plasmid Preparation, Software