Dynamic competition between a ligand and transcription factor NusA governs riboswitch-mediated transcription regulation

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.

Observation of coordinated cotranscriptional RNA folding events

“…The benefits of this approach are evident in our reinvestigation of ZTP and fluoride riboswitch folding and our analysis of ppGpp riboswitch folding: in all cases, improved resolution of 3' proximal RNA structures enabled the detection of cotranscriptional folding events in which coordinated reactivity changes spanned the entire transcript. In combination with complementary biophysical approaches [8][9][10][11][12][13] , the ability to resolve coordinated structural changes by cotranscriptional RNA chemical probing will likely aid efforts to predict RNA folding pathways [40][41][42][43][44][45][46] .…”

“…In the past several years, the development of methods that can monitor cotranscriptional RNA structure formation with high temporal and spatial resolution has rapidly advanced our ability to characterize cotranscriptional RNA folding mechanisms. These complementary approaches include applications of single-molecule force spectroscopy 8,9 and single-molecule FRET [10][11][12][13] , which measure cotranscriptional RNA folding with high temporal resolution, and the application of high-throughput RNA chemical probing to map the structure of cotranscriptionally folded intermediate transcripts at nucleotide resolution [14][15][16] .…”

Mechanism of Mg2+ Mediated Encapsulation of an Anionic Cognate Ligand in a Bacterial Riboswitch

“…For the riboswitch to control the transcription process, an interplay of the transcription speed, the folding time of the aptamer domain as the polymerase synthesizes it, and ligand binding to the aptamer domain are critical in determining the outcome of gene regulation. 28,29,62 An exciting extension of this work would be to study the co-transcriptional folding of the fluoride riboswitch 28,29,63 to explore at what stage of the aptamer domain synthesis does the fluoride ion binds and bifurcates the folding pathways, which either promote or inhibit the transcription terminator helix formation. However, this will be an extremely challenging computation.…”

Tuning Strand Displacement Kinetics Enables Programmable ZTP Riboswitch Dynamic Rangein vivo

“…Early work demonstrated that transcriptional riboswitch function is highly dependent upon the rate of transcription (NTP concentration) and transcriptional dynamics (RNAP pausing) (9), but mechanistic details of how EPs fold during the kinetic regime of transcription have been harder to decipher. Recent advances applying diverse biophysical techniques and RNA structure chemical probing methods are beginning to uncover some of these cotranscriptional EP folding mechanisms (10–16). Collectively, these studies are starting to show that many transcriptional ON-riboswitches perform rapid decision-making between two mutually exclusive EP structural states via internal strand displacement mechanisms.…”