The conformational landscape of transcription intermediates involved in the regulation of the ZMP-sensing riboswitch from Thermosinus carboxydivorans

Abstract

<jats:title>Abstract</jats:title> <jats:p>Recently, prokaryotic riboswitches have been identified that regulate transcription in response to change of the concentration of secondary messengers. The ZMP (5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR))-sensing riboswitch from Thermosinus carboxydivorans is a transcriptional ON-switch that is involved in purine and carbon-1 metabolic cycles. Its aptamer domain includes the pfl motif, which features a pseudoknot, impeding rho-independent terminator formation upon stabilization by ZMP interaction. We herein investigate the conformational landscape of transcriptional intermediates including the expression platform of this riboswitch and characterize the formation and unfolding of the important pseudoknot structure in the context of increasing length of RNA transcripts. NMR spectroscopic data show that even surprisingly short pre-terminator stems are able to disrupt ligand binding and thus metabolite sensing. We further show that the pseudoknot structure, a prerequisite for ligand binding, is preformed in transcription intermediates up to a certain length. Our results describe the conformational changes of 13 transcription intermediates of increasing length to delineate the change in structure as mRNA is elongated during transcription. We thus determine the length of the key transcription intermediate to which addition of a single nucleotide leads to a drastic drop in ZMP affinity.</jats:p>

Real-time monitoring of single ZTP riboswitches reveals a complex and kinetically controlled decision landscape

“…1), suggesting that even partial termination hairpin folding competes with ligand binding. Consistent with a recent study on the Thermosinus carboxydivorans ZTP riboswitch 26 , these measurements suggest that, at equilibrium, there should exist an~10 nt ligand binding window. We labeled Δterm with donor (Cy3) and acceptor (Cy5) fluorophores 27 and tested for a ZMP-responsive conformational change using single-molecule FRET 28,29 (Fig.…”

Parallel Discovery Strategies Provide a Basis for Riboswitch Ligand Design

“…As the F. ulcerans ZTP riboswitch is an ''ON'' switch, adding increasing ZMP to the transcription reaction stabilizes the aptamer fold of the RNA, prevents termination, and leads to the accumulation of the readthrough product (Figure 3B), which can be quantified and fit to determine the concentration at which the riboswitch is half activated (T 50 ). In these experiments, T 50 values are higher than K d values determined from other methods, as the riboswitch is kinetically controlled under these conditions (Binas et al, 2020;Strobel et al, 2019;Wickiser et al, 2005b). Thus, the T 50 values are non-equilibrium measurements that reflect ligand binding on rates (k on ).…”

Tuning Strand Displacement Kinetics Enables Programmable ZTP Riboswitch Dynamic Rangein vivo

“…Previous work with the Clostridium beijerinckii (Cbe) pfl ZTP riboswitch showed that this disruption of the AD in the absence of Z proceeds via a strand displacement mechanism (15), which has been supported by later smFRET and NMR studies of the Fusobacterium ulcerans and Thermosinus carboxydivorans ZTP riboswitches, respectively (13,30). Building off of this work, we used a functional mutagenesis approach with E. coli in vivo reporter assays to show that kinetic barriers to strand displacement can be used to tune ZTP riboswitch dynamic range.…”