Work Description

Date: 09 October, 2023

Dataset Title: Single-molecule microscopy image data and analysis files for "Single-molecule FRET observes opposing effects of urea and TMAO on structurally similar meso- and thermophilic riboswitch RNAs"

Dataset Creators: Q. Hou, S. Chatterjee, P.E. Lund, K.C. Suddala, N.G. Walter

Dataset Contact: Qian Hou houq@umich.edu

Key Points:
- We compared the conformational dynamics of structurally similar Bacillus subtilis (Bsu) and Thermoanaerobacter tengcongensis (Tte) preQ1 riboswitches using single-molecule fluorescence resonance energy transfer (smFRET) microscopy.
- We generated data showing the changes in the structural compactness of the two riboswitches in response to the perturbation of urea, trimethylamine N -oxide (TMAO) and divalent metal ion Mg2+.
- We generated kinetic data showing the changes in folding rate constants in response to the presence of urea, TMAO and Mg2+.
- Finally, our m-value analysis of the two riboswitches shows their performance in resisting chemical perturbation.

Research Overview:
Bacteria live in a broad range of environmental temperatures that require adaptations of their RNA sequences to maintain function. Riboswitches are regulatory RNAs that change conformation upon binding of typical metabolite ligands to control bacterial gene expression. The paradigmatic small class-I preQ1 riboswitches from the mesophile Bacillus subtilis (Bsu) and the thermophile Thermoanaerobacter tengcongensis (Tte) adopt similar pseudoknot structures when bound to preQ1. Here, we use UV-melting analysis combined with single-molecule detected chemical denaturation by urea to compare the thermodynamic and kinetic folding properties of the two riboswitches, and the urea-countering effects of trimethylamine N-oxide (TMAO). Our results show that, first, the Tte riboswitch is more thermotolerant than the Bsu riboswitch, despite only subtle sequence differences. Second, using single-molecule FRET, we find that urea destabilizes the folded pseudoknot structure of both riboswitches, yet has a lower impact on the unfolding kinetics of the thermodynamically less stable Bsu riboswitch. Third, our analysis shows that TMAO counteracts urea denaturation and promotes folding of both the riboswitches, albeit with a smaller effect on the more stable Tte riboswitch. Together, these findings elucidate how subtle sequence adaptations in a thermophilic bacterium can stabilize a common RNA structure when a new ecological niche is conquered.

Methodology:
Single-Molecule Fluorescence Resonance Energy Transfer (smFRET) studies were conducted on a prism-based total internal reflection fluorescence (TIRF) microscope. We used RNA riboswitch molecules, doubly labeled with DY547 and Cy5, to monitor their molecular conformational dynamics and the associated changes in smFRET values. Using a 532 nm laser, DY547 was excited, and emissions from both DY547 and Cy5 were simultaneously captured with an intensified charge-coupled device camera (ICCD, I-Pentamax, Princeton Instruments) set at a 60 ms time resolution. We processed the captured footage using IDL (Research Systems) to extract smFRET time trajectories, which were subsequently assessed using MATLAB (The Math Works) scripts. We manually selected smFRET time trajectories that met the following criteria: single-step photobleaching, a trajectory duration of at least 100 camera frames before bleaching, a signal-to-noise ratio greater than 4:1, and a total fluorescence intensity (donor + acceptor) exceeding 300 (in arbitrary units). FRET efficiency was defined as EFRET = IA/(ID + IA). Here, ID and IA represent the background-corrected intensity levels of DY547 (donor) and Cy5 (acceptor) fluorophores. Dynamic time traces were idealized using a two-state model according to Hidden-Markov Modeling (HMM) and the segmental k-means algorithm in the QuB software suite (State University of New York at Buffalo). FRET efficiencies were further analyzed to generate 1) FRET distribution histograms by combining the first 100 frames from each time trace using MATLAB, 2) Transition Density Plots (TDPs) and Transition Occupancy Density Plots (TODPs) using MATLAB, and 3) rate constants kdock and kundock by extracting dwell times in the docked and undocked states from all the idealized traces and fitting the cumulative dwell time distributions to exponential functions.

Instrument and/or Software specifications:
- Prism-based total internal reflection fluorescence (TIRF) microscope
- Intensified charge-coupled device camera (ICCD, I-Pentamax, Princeton Instruments)
- IDL (Research Systems)
- MATLAB (The Math Works)
- QuB software suite (State University of New York at Buffalo)

Files contained here:
This data includes the experimental findings and associated analyses detailed in the research article titled "Single-molecule FRET observes opposing effects of urea and TMAO on structurally similar meso- and thermophilic riboswitch RNAs". The data consists of multiple zip files, each representing an experiment that corresponds to the key results in the publication. Each experiment includes movies, qualifying smFRET trajectories, and analysis files related to various conditions within that experimental group.

This data is composed of ten experimental conditions, which is described below:
01 Bsu preQ1 riboswitch, 100 nM preQ1 ligand, urea titration
02 Bsu preQ1 riboswitch, 100 nM preQ1 ligand, 1 mM, Mg2+, urea titration
03 Tte preQ1 riboswitch, 100 nM preQ1 ligand, urea titration
04 Tte preQ1 riboswitch, 100 nM preQ1 ligand, 1 mM, Mg2+, urea titration
05 Bsu preQ1 riboswitch, 100 nM preQ1 ligand, TMAO titration
06 Bsu preQ1 riboswitch, 100 nM preQ1 ligand, 1 mM, Mg2+, TMAO titration
07 Bsu preQ1 riboswitch, 100 nM preQ1 ligand, 2 M urea, TMAO titration
08 Bsu preQ1 riboswitch, 100 nM preQ1 ligand, 1 mM, Mg2+, 2 M urea, TMAO titration
09 Tte preQ1 riboswitch, 100 nM preQ1 ligand, 2 M urea, TMAO titration
10 Tte preQ1 riboswitch, 100 nM preQ1 ligand, 1 mM, Mg2+, 2 M urea, TMAO titration

DG16: data compiled from m-value analysis
This file compiles all the calculations for the m-value analysis.
- deltaG_fold = -RTlnKeq = -RTln(AUC_hiFRET/AUC_midFRET)
- R = the gas constant
- T is the temperature on the Kelvin scale.
- lambda2 value = AUC_hiFRET
- lambda1 value = AUC_midFRET
- Error was generated by bootstrapping.

DG18: kinetic rate constants compiled from kinetic analysis
This file compiles the rate constants extracted from condition 1 to 10.
- k(dock) is the folding rate constant.
- k(undock) is the unfolding rate constant.
- Total traces are the total amount of FRET traces which fulfill our manual selection criteria.
- N kinetic traces are traces with kinetic features.
- Error was generated by bootstrapping.

** Within each experimental condition folder, the data can be grouped into three categories: 1) raw microscope data 2) data selected for conformational analysis (histogram traces) 3) data selected for kinetic analysis (kinetic traces).
** Here is an example explaining the folder structure for each experimental condition.
1) e.g., 0M_urea
Within this folder, you will find the original raw microscope movie files.
2) e.g., histogram_traces_0M_urea
Within this folder, you will find the traces selected to generate histogram plots. You will also find the bootstrapping results used to generate error bars.
* histogram_traces_0M_urea > RemNegOut > QuB > parsed
Within this folder, you will find the TODP data.
3) e.g., kinetics_traces_0M_urea
Within this folder, you will find the traces selected for calculating rate constants.
* kinetics_traces_0M_urea > RemNegOut > QuB_faster rate_22Feb16 > parsed
Within this folder, you will find the TDP data, rate constant fittings and bootstrapping data.

Related publication(s):
Hou, Q., et al. (2023). Single-molecule FRET observes opposing effects of urea and TMAO on structurally similar meso- and thermophilic riboswitch RNAs. https://doi.org/10.1093/nar/gkad866

Use and Access:
This data set is made available under a Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) lisence.

To Cite Data:
Hou, Q., Chatterjee, S., Lund, P. E., Suddala, K. C., Walter, N. G. Single-molecule microscopy image data and analysis files for "Single-molecule FRET observes opposing effects of urea and TMAO on structurally similar meso- and thermophilic riboswitch RNAs" [Data set], University of Michigan - Deep Blue Data.