Transcriptional pausing coordinates folding of the aptamer domain and the expression platform of a riboswitch

Abstract

Riboswitches are cis-acting elements that regulate gene expression by affecting transcriptional termination or translational initiation in response to binding of a metabolite. A typical riboswitch is made of an upstream aptamer domain and a downstream expression platform. Both domains participate in the folding and structural rearrangement in the absence or presence of its cognate metabolite. RNA polymerase pausing is a fundamental property of transcription that can influence RNA folding. Here we show that pausing plays an important role in the folding and conformational rearrangement of the Escherichia coli btuB riboswitch during transcription by the E. coli RNA polymerase. This riboswitch consists of an approximately 200 nucleotide, coenzyme B12 binding aptamer domain and an approximately 40 nucleotide expression platform that controls the ribosome access for translational initiation. We found that transcriptional pauses at strategic locations facilitate folding and structural rearrangement of the full-length riboswitch, but have minimal effect on the folding of the isolated aptamer domain. Pausing at these regulatory sites blocks the formation of alternate structures and plays a chaperoning role that couples folding of the aptamer domain and the expression platform. Pausing at strategic locations may be a general mechanism for coordinated folding and conformational rearrangements of riboswitch structures that underlie their response to environmental cues.gene regulation | translational control | metabolism R NA structures fulfill important roles in the regulation of gene expression including the control of translational initiation, transcriptional termination, and alternative splicing (1-5). In many cases, an RNA conformational change is crucial to gene regulation since one RNA sequence may adopt alternate structures. Regulation of gene expression is achieved when an input domain senses varying cellular conditions, resulting in shifting the balance between two or more structural states.A prime example of gene regulation by RNA conformational changes in bacteria is riboswitch control. Riboswitches are cisacting elements that regulate gene expression in response to the concentration of an intracellular metabolite. The coenzyme B12 riboswitch is located in the 5′ untranslated region of the btuB gene which encodes an outer membrane transporter for B12 (6) (Fig. 1A). The btuB riboswitch consists of two domains: an upstream coenzyme B12 binding aptamer and a downstream expression platform. When the cellular concentration of coenzyme B12 is high, this metabolite binds to the aptamer, reconfiguring the expression platform to form a structure in which the ribosome binding site (RBS) is base paired and inaccessible. When the concentration of coenzyme B12 is low, the mRNA forms an alternate structure which allows for translation (6, 7).RNA folding and conformational switching occurs during transcription in vivo. Folding during transcription is particularly important for riboswitch regulation (8). Although bacterial RNA ...

Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution

“…2C, Lower Center). Saturating concentrations of TPP (100 μM) (19,22) further stabilized the P1 helix. Here, TPP's predominant impact was to reduce residual dynamics in the system, suggesting a further stabilization of the P1 helix (Fig.…”

“…3). We hypothesize that such behaviors arise from relatively slow conformational changes [also observed in other riboswitch systems (19,33)] within the core domain of the TPP aptamer within the J2-4 junction and related distortions in the RNA backbone at the union of the P4 and P5 helices (Fig. 1).…”

“…In this context, it is important to note that RNA polymerase has been shown to pause immediately after synthesis of the E. coli thiM TPP aptamer within the expression platform at the sequence surrounding the ribosome-binding site (20). As pausing of this nature would provide time for the equilibration of alternative folds and TPP sensing, the thiM riboswitch, like other riboswitch domains (9,19,20,41), is likely to exhibit aspects of both kinetic and thermodynamic control (9,42). The observed instability of the TPP aptamer fold and helix P1-in both the absence and the presence of saturating concentrations of the TPP ligand-also suggests that the thiM-mediated regulatory outcome during translation initiation may also be strongly influenced by sequence modifications and environmental variables (temperature and ionic conditions) and the extent to which conformational selection and adaptive-or induced-fit-processes (43)(44)(45)(46) contribute to ligand recognition.…”

“…Consequently, little is presently known about the determinants of alternative riboswitch folding pathways and how ligands regulate these events. A deeper understanding of both bound and unbound forms of the aptamer and expression platforms is required to gain mechanistic insights into the regulatory switch that they induce (9,(18)(19)(20). The nature and timing of the foldingrecognition process in riboswitches imply potentially complex and rapid dynamic processes within the nascent RNA chain (21).…”

Disparity between microRNA levels and promoter strength is associated with initiation rate and Pol II pausing

“…The lengthy pausing calculated here is consistent with the reported slow kinetics of co-transcriptional miRNA processing by the microprocessor 5 . Pausing at the pre-miRNA may be important for its appropriate folding, reminiscent of the pausing at riboswitches of bacteria, which was proposed to provide a time window for the RNA to fold without interference of competing sequences present downstream 46,47 .…”