Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation

Abstract

On the basis of nascent transcript sequencing, it has been postulated but never demonstrated that transcriptional pausing at translation start sites is important for gene regulation. Here we show that the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch contains a regulatory pause site in the translation initiation region that acts as a checkpoint for thiC expression. By biochemically probing nascent transcription complexes halted at defined positions, we find a narrow transcriptional window for metabolite binding, in which the downstream boundary is delimited by the checkpoint. We show that transcription complexes at the regulatory pause site favour the formation of a riboswitch intramolecular lock that strongly prevents TPP binding. In contrast, cotranscriptional metabolite binding increases RNA polymerase pausing and induces Rho-dependent transcription termination at the checkpoint. Early transcriptional pausing may provide a general mechanism, whereby transient transcriptional windows directly coordinate the sensing of environmental cues and bacterial mRNA regulation.

Requirements for efficient cotranscriptional regulatory switching in designed variants of the Bacillus subtilis pbuE adenine-responsive riboswitch

“…A second feature of the nucleator element that may be important for efficient regulation is a potential "programmed pause" at the 3'-side of the helix. It well established that many riboswitches contain stretches of uridines in their expression platforms that represent RNA polymerase pause sites that give more time for events such as RNA folding and ligand binding to occur [13,17,49,50]. In the pbuE riboswitch, a hexa-uridine tract is located at the 3'-side of the nucleator element that could serve as a pause site [13].…”

“…One major hurdle in the robust design of riboswitches is that they generally only function in the context of transcription [1,[12][13][14], requiring careful consideration of kinetic processes such as rates of ligand binding, higher-order RNA folding and secondary structural switching processes such as strand exchange [15]. Furthermore, the influence of RNA sequence on processes such as transcriptional pausing and participation of factors such as NusA or Rho on riboswitches in the cellular context further confounds their design and implementation [16][17][18]. It is likely that many of these processes are influenced by cryptic sequence elements difficult to engineer into an RNA of interest.…”

Chemical transcription roadblocking for nascent RNA display

“…Nascent RNA display is a powerful tool that has proven applications in mapping RNA structure and folding (1)(2)(3)(4) and systematic characterization of RNA function (5,6). Established approaches for halting RNAP have typically relied on protein roadblocks that are not easily navigable by transcribing polymerases.…”

“…The in vitro display of nascent RNA molecules from halted transcription elongation complexes is a powerful tool with proven applications in nascent RNA folding (1)(2)(3)(4) and systematic RNA aptamer characterization (5,6). Historically, transcription arrest at a defined DNA coordinate has been achieved by attaching a protein roadblock to a DNA template to halt the progression of RNA polymerases (RNAPs).…”

Applications and limitations of regulatoryRNAelements in synthetic biology and biotechnology

“…One kinetic model suggests that the speed of transcription can determine riboswitch function (Wickiser et al 2005). Another study suggests that transcriptional pausing at the translational start site is necessary, during transcription, for the aptamer to fold into its functional structure before the ligand can bind (Chauvier et al 2017). During this pause or lag, the metabolite is waiting to access the newly transcribed aptamer.…”