Photoaffinity labeling of 23 S rRNA in<i>Escherichia coli</i> ribosomes with poly(U)‐coded ethyl 2‐diazomalonyl‐Phe‐tRNA

Bispink, L.; Matthaei, Heinrich

doi:10.1016/0014-5793(73)80480-6

Cited by 55 publications

(1 citation statement)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Early pioneering studies demonstrated the broad potential of (photo)chemical methods to investigate nucleic acid structure and function. − An emerging approach to study RNA is applying small chemical probes that bind RNA with high affinity and that form covalent bonds between the probe and RNA of interest. The covalently bound small-molecule probe can be subsequently modified for analysis.…”

Section: Introductionmentioning

confidence: 99%

Affinity-Based Profiling of the Flavin Mononucleotide Riboswitch

et al. 2022

View full text Add to dashboard Cite

Riboswitches are structural RNA elements that control gene expression. These naturally occurring RNA sensors are of continued interest as antibiotic targets, molecular sensors, and functional elements of synthetic circuits. Here, we describe affinity-based profiling of the flavin mononucleotide (FMN) riboswitch to characterize ligand binding and structural folding. We designed and synthesized photoreactive ligands and used them for photoaffinity labeling. We showed selective labeling of the FMN riboswitch and used this covalent interaction to quantitatively measure ligand binding, which we demonstrate with the naturally occurring antibiotic roseoflavin. We measured conditional riboswitch folding as a function of temperature and cation concentration. Furthermore, combining photoaffinity labeling with reverse transcription revealed ligand binding sites within the aptamer domain with single-nucleotide resolution. The photoaffinity probe was applied to cellular extracts of Bacillus subtilis to demonstrate conditional folding of the endogenous low-abundant ribD FMN riboswitch in biologically derived samples using quantitative PCR. Lastly, binding of the riboswitch-targeting antibiotic roseoflavin to the FMN riboswitch was measured in live bacteria using the photoaffinity probe.

show abstract