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Updated: Oct 13, 2025

In Vitro Transcription Assays and Their Application in Drug Discovery
Published on: September 20, 2016
Dynamic competition between a ligand and transcription factor NusA governs riboswitch-mediated transcription
Adrien Chauvier1,2, Pujan Ajmera1,2, Rajeev Yadav1,2
1Single Molecule Analysis Group, University of Michigan, Ann Arbor, MI 48109.
Abstract:
Cotranscriptional RNA folding is widely assumed to influence the timely control of gene expression, but our understanding remains limited. In bacteria, the fluoride (F-)-sensing riboswitch is a transcriptional control element essential to defend against toxic F- levels. Using this model riboswitch, we find that its ligand F- and essential bacterial transcription factor NusA compete to bind the cotranscriptionally folding RNA, opposing each other's modulation of downstream pausing and termination by RNA polymerase. Single-molecule fluorescence assays probing active transcription elongation complexes discover that NusA unexpectedly binds highly reversibly, frequently interrogating the complex for emerging, cotranscriptionally folding RNA duplexes. NusA thus fine-tunes the transcription rate in dependence of the ligand-responsive higher-order structure of the riboswitch. At the high NusA concentrations found intracellularly, this dynamic modulation is expected to lead to adaptive bacterial transcription regulation with fast response times.
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