A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration.

Widespread manganese-sensing transcriptional riboswitches effect the dependable gene regulation needed for bacterial manganese homeostasis in changing environments. Riboswitches - like most structured RNAs - are believed to fold co-transcriptionally, subject to both ligand binding and transcription events; yet how these processes are orchestrated for robust regulation is poorly understood. Through a combination of single-molecule and bulk approaches, we discover how a single Mn2+ ion and the transcribing RNA polymerase (RNAP), paused immediately downstream by a DNA template sequence, are coordinated by the bridging switch helix P1.1 in the representative Lactococcus lactis riboswitch. This coordination achieves a heretofore-overlooked semi-docked global conformation of the nascent RNA, P1.1 base pair stabilization, transcription factor NusA ejection, and RNAP pause extension, thereby enforcing transcription readthrough. Our work demonstrates how a central, adaptable RNA helix functions analogous to a molecular fulcrum of a first-class lever system to integrate disparate signals for finely balanced gene expression control.

Looking at Biomolecular Interactions through the Lens of Correlated Fluorescence Microscopy and Optical Tweezers.

Understanding complex biological events at the molecular level paves the path to determine mechanistic processes across the timescale necessary for breakthrough discoveries. While various conventional biophysical methods provide some information for understanding biological systems, they often lack a complete picture of the molecular-level details of such dynamic processes. Studies at the single-molecule level have emerged to provide crucial missing links to understanding complex and dynamic pathways in biological systems, which are often superseded by bulk biophysical and biochemical studies. Latest developments in techniques combining single-molecule manipulation tools such as optical tweezers and visualization tools such as fluorescence or label-free microscopy have enabled the investigation of complex and dynamic biomolecular interactions at the single-molecule level. In this review, we present recent advances using correlated single-molecule manipulation and visualization-based approaches to obtain a more advanced understanding of the pathways for fundamental biological processes, and how this combination technique is facilitating research in the dynamic single-molecule (DSM), cell biology, and nanomaterials fields.

Precise tuning of bacterial translation initiation by non-equilibrium 5'-UTR unfolding observed in single mRNAs.

Noncoding, structured 5'-untranslated regions (5'-UTRs) of bacterial messenger RNAs (mRNAs) can control translation efficiency by forming structures that either recruit or repel the ribosome. Here we exploit a 5'-UTR embedded preQ1-sensing, pseudoknotted translational riboswitch to probe how binding of a small ligand controls recruitment of the bacterial ribosome to the partially overlapping Shine-Dalgarno (SD) sequence. Combining single-molecule fluorescence microscopy with mutational analyses, we find that the stability of 30S ribosomal subunit binding is inversely correlated with the free energy needed to unfold the 5'-UTR during mRNA accommodation into the mRNA binding cleft. Ligand binding to the riboswitch stabilizes the structure to both antagonize 30S recruitment and accelerate 30S dissociation. Proximity of the 5'-UTR and stability of the SD:anti-SD interaction both play important roles in modulating the initial 30S-mRNA interaction. Finally, depletion of small ribosomal subunit protein S1, known to help resolve structured 5'-UTRs, further increases the energetic penalty for mRNA accommodation. The resulting model of rapid standby site exploration followed by gated non-equilibrium unfolding of the 5'-UTR during accommodation provides a mechanistic understanding of how translation efficiency is governed by riboswitches and other dynamic structure motifs embedded upstream of the translation initiation site of bacterial mRNAs.

A translational riboswitch coordinates nascent transcription-translation coupling.

Bacterial messenger RNA (mRNA) synthesis by RNA polymerase (RNAP) and first-round translation by the ribosome are often coupled to regulate gene expression, yet how coupling is established and maintained is ill understood. Here, we develop biochemical and single-molecule fluorescence approaches to probe the dynamics of RNAP-ribosome interactions on an mRNA with a translational preQ1-sensing riboswitch in its 5' untranslated region. Binding of preQ1 leads to the occlusion of the ribosome binding site (RBS), inhibiting translation initiation. We demonstrate that RNAP poised within the mRNA leader region promotes ribosomal 30S subunit binding, antagonizing preQ1-induced RBS occlusion, and that the RNAP-30S bridging transcription factors NusG and RfaH distinctly enhance 30S recruitment and retention, respectively. We further find that, while 30S-mRNA interaction significantly impedes RNAP in the absence of translation, an actively translating ribosome promotes productive transcription. A model emerges wherein mRNA structure and transcription factors coordinate to dynamically modulate the efficiency of transcription-translation coupling.

Transcriptional Riboswitches Integrate Timescales for Bacterial Gene Expression Control.

Transcriptional riboswitches involve RNA aptamers that are typically found in the 5' untranslated regions (UTRs) of bacterial mRNAs and form alternative secondary structures upon binding to cognate ligands. Alteration of the riboswitch's secondary structure results in perturbations of an adjacent expression platform that controls transcription elongation and termination, thus turning downstream gene expression "on" or "off." Riboswitch ligands are typically small metabolites, divalent cations, anions, signaling molecules, or other RNAs, and can be part of larger signaling cascades. The interconnectedness of ligand binding, RNA folding, RNA transcription, and gene expression empowers riboswitches to integrate cellular processes and environmental conditions across multiple timescales. For a successful response to an environmental cue that may determine a bacterium's chance of survival, a coordinated coupling of timescales from microseconds to minutes must be achieved. This review focuses on recent advances in our understanding of how riboswitches affect such critical gene expression control across time.

Local-to-global signal transduction at the core of a Mn2+ sensing riboswitch.

The widespread Mn2+-sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch aptamer from Xanthomonas oryzae at 2.96 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >100 µs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.