An anionic ligand snap-locks a long-range interaction in a magnesium-folded riboswitch

The archetypical transcriptional crcB fluoride riboswitch from Bacillus cereus is an intricately structured non-coding RNA element enhancing gene expression in response to toxic levels of fluoride. Here, we used single molecule FRET to uncover three dynamically interconverting conformations appearing along the transcription process: two distinct undocked states and one pseudoknotted docked state. We find that the fluoride anion specifically snap-locks the magnesium-induced, dynamically docked state. The long-range, nesting, single base pair A40-U48 acts as the main linchpin, rather than the multiple base pairs comprising the pseudoknot. We observe that the proximally paused RNA polymerase further fine-tunes the free energy to promote riboswitch docking. Finally, we show that fluoride binding at short transcript lengths is an early step toward partitioning folding into the docked conformation. These results reveal how the anionic fluoride ion cooperates with the magnesium-associated RNA to govern regulation of downstream genes needed for fluoride detoxification of the cell.

The many states of STIM1

Harnessing single-molecule FRET illuminates the structural changes necessary for a protein to fine-tune the influx of calcium when reserves inside a cell run low.

Kinetics of DNA looping by Anabaena sensory rhodopsin transducer (ASRT) by using DNA cyclization assay

DNA cyclization assay together with single-molecule FRET was employed to monitor protein-mediated bending of a short dsDNA (~ 100 bp). This method provides a simple and easy way to monitor the structural change of DNA in real-time without necessitating prior knowledge of the molecular structures for the optimal dye-labeling. This assay was applied to study how Anabaena sensory rhodopsin transducer (ASRT) facilitates loop formation of DNA as a possible mechanism for gene regulation. The ASRT-induced DNA looping was maximized at 50 mM of Na+, while Mg2+ also played an essential role in the loop formation.

Inferring kinetic rate constants from single-molecule FRET trajectories – a blind benchmark of kinetic analysis tools

Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We tested them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

Molecular basis of ubiquitin-specific protease 8 autoinhibition by the WW-like domain

Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in multiple membrane trafficking pathways. The enzyme activity is inhibited by binding to 14-3-3 proteins. Mutations in the 14-3-3-binding motif in USP8 are related to Cushing’s disease. However, the molecular basis of USP8 activity regulation remains unclear. This study identified amino acids 645–684 of USP8 as an autoinhibitory region, which might interact with the catalytic USP domain, as per the results of pull-down and single-molecule FRET assays performed in this study. In silico modelling indicated that the region forms a WW-like domain structure, plugs the catalytic cleft, and narrows the entrance to the ubiquitin-binding pocket. Furthermore, 14-3-3 inhibited USP8 activity partly by enhancing the interaction between the WW-like and USP domains. These findings provide the molecular basis of USP8 autoinhibition via the WW-like domain. Moreover, they suggest that the release of autoinhibition may underlie Cushing’s disease due to USP8 mutations.