Beyond Ligand Binding: Single Molecule Observation Reveals How Riboswitches Integrate Multiple Signals to Balance Bacterial Gene Regulation

Abstract

Riboswitches are specialized RNA structures that orchestrate gene expression in response to sensing specific metabolite or ion ligands, mostly in bacteria. Upon ligand binding, these conformationally dynamic RNA motifs undergo structural changes that control critical gene expression processes such as transcription termination and translation initiation, thereby enabling cellular homeostasis and adaptation. Because RNA folds rapidly and co-transcriptionally, riboswitches make use of the low complexity of RNA sequences to adopt alternative, transient conformations on the heels of the transcribing RNA polymerase (RNAP), resulting in kinetic partitioning that defines the regulatory outcome. This review summarizes single molecule microscopy evidence that has begun to unveil a sophisticated network of dynamic, kinetically balanced interactions between riboswitch architecture and the gene expression machinery that, together, integrate diverse cellular signals.

Introduction

Bacterial riboswitches are versatile RNA regulatory elements that modulate gene expression through their ability to bind small molecules such as metabolites, elemental ions, or other RNAs [1]. These noncoding RNA (ncRNA) sequences are located in the 5'-untranslated regions (5'-UTRs) of messenger RNAs (mRNA) and can alter their secondary and tertiary structures upon ligand binding. The structural rearrangements serve as a molecular switch to activate or repress target gene expression by affecting essential processes such as transcription termination, initiation of translation, and RNA degradation (Figure 1) (reviewed in [2]).

Figure 1.

Riboswitches regulate essential biological processes. Riboswitches are RNA structural elements that change conformation upon binding a specific metabolite or ion (termed ligand), which in turn allows for the modulation of gene expression at the level of mRNA transcription (transcriptional) (a) or translation initiation (translational) (b). SD = Shine Dalgarno sequence.

As they are synthesized from the start of the gene by RNA polymerase (RNAP), riboswitches dynamically adopt transient structures that can decisively influence their regulatory outcomes. The nature of RNA folding (typically on the microsecond timescale for local structures and close-range interactions, faster than the millisecond timescale of nucleotide incorporation) allows them to fold co-transcriptionally, which enables a rapid, adaptive adjustment to changing cellular environments directly in the wake of RNAP progression [2,3]. Due to the fleeting and context-dependent nature of these interconverting structures – made possible by the relatively low sequence complexity of RNA that allows for a multitude of alternative base pairings of near-equal thermodynamic stability – conventional, ensemble-averaging bulk methods often fall short in capturing the nuances of their formation and function.

Single-molecule techniques have emerged as exceptionally well-suited tools for disentangling the complexities of riboswitch regulation (reviewed in [4]). These advanced methodologies provide unparalleled insights into the real-time dynamics of RNA folding and ligand interaction at the single molecule level. By observing riboswitch behavior one molecule at a time, researchers can avoid ensemble averaging, thereby measuring complex and transient kinetics, and gaining a more authentic view of the diverse regulatory states and pathways that define the functional landscapes of riboswitches. Such detailed observations are essential for a mechanistic understanding of how these RNA sensors integrate multiple signals to control gene regulatory outcomes, offering promising avenues to explore and therapeutically exploit the intricacies of RNA-based regulation [5].

Using single molecule techniques to characterize the dynamic properties of riboswitches, we examine recent key observations that are reshaping our understanding of co-transcriptional folding mechanisms. These approaches are providing unprecedented insights into the temporal and structural intricacies of riboswitch function during the nascent phase of transcription, yielding unique scientific perspectives on their role as integrators of cellular signals. As we delve into the latest findings, we explore the implications of co-transcriptional folding for gene regulation and the broader landscape of RNA biology within the context of the bacterial cell.

Regulatory Checkpoints during Transcription

The meticulous study of RNA co-transcriptional folding in real-time is key to unraveling the sophisticated mechanisms by which riboswitches, representative of many other structured RNAs, modulate gene expression [2]. A paramount aspect of this regulation is the formation of transient RNA structures that emerge temporarily during the transcription process [6,7]. These fleeting intermediates, some of which are longer-lived and serve as kinetic traps, can critically dictate the pathway of RNA folding, dynamically respond to changes in cellular conditions of a rapidly dividing bacterium exposed to environmental pressures, and subsequently influence the outcome of the gene response [8-10] (Figure 2).

Figure 2.

Transcription checkpoints determine the fate of riboswitch-mediated regulation of gene expression. During transcription, ligand binding efficiency increases progressively as the RNAP reaches the regulatory checkpoint pause site, promoting the adoption of a binding-competent aptamer conformation. The ligand concentration dependent binding and ligand dissociation rate constants relative to those of transcription pause escape then determine the partitioning between the ligand-free and ligand-bound pathways (Sensing Window). In the expression platform, ligand binding thermodynamically stabilizes a structure that either promotes or prevents downstream gene expression. In the ligand-free conformation, transcription speed and RNA folding rate are in direct competition, effecting kinetically controlled partitioning (Decision Window).

Pioneering ensemble-averaging probing methodologies, such as co-transcriptional SHAPE-seq, paved the way for observing the sequence of riboswitch folding events as it is transcribed one nucleotide at a time [11,12]. Accordingly, the transient states of a riboswitch can be isolated and monitored with precision, mapping how the folding intermediates interrelate with functional outcomes. A critical process within this folding landscape is the recent allusion to the ‘strand invasion’ or ‘strand displacement’ mechanism, whereby distinct RNA structures exchange base pair by base pair with low energy barrier and thermodynamic cost [13]. Strand invasion is characterized by a competitive folding scenario where an RNA strand transiently pairs with complementary bases, often interrupting or preventing the formation of more stable secondary structures (reviewed in [14]). Notably, the DNA nanotechnology field has long used closely related toehold mediated strand displacement reactions for very similar purposes[15]. This competition becomes particularly crucial in the context of transcriptionally acting riboswitches in which the formation of a terminator stem structure plays a pivotal role in signaling the end of transcription. While these terminator stems are typically thermodynamically stable and promote transcription termination [16], they are challenged by alternative, less stable RNA structures that can form earlier during transcription elongation and sequester some of the same nucleotides by base pairing them with upstream sequences (Figure 2).

At the single molecule (or single transcript) level, a specific transient conformation has been observed for the thiamin pyrophosphate (TPP) sensing riboswitch, termed 'anti-P1', and has emerged as a standout character in the folding sequence [17,18]. The elusive anti-P1 structure forms during transcription and significantly impacts ligand binding. It was observed that once the anti-P1 structure is established, especially during transcriptional pauses, ligand binding is hindered. This so-called ‘transcriptional checkpoint’ presents a dramatic account of how the temporal aspects of transcription - specifically, RNAP pausing - can intercede, promoting the formation of a transient structure and thereby preventing the riboswitch from assuming its repressive conformation in the ligand-bound state (Figure 2). Similar observations have been made for an adenine-sensing riboswitch, where an upstream, alternative secondary structure is adopted in a temperature-dependent context that renders the bacterium temperature-compensatory in its gene regulatory response [19].

Bacterial polymerases are well known to display uneven speeds as they transcribe a DNA template, allowing regulatory mechanisms to further modulate elongation rate or affect termination[10,16]. Sequence signals in the non-template DNA strand, binding of transcription factors, and secondary structure elements of the nascent RNA have all been found to have such regulatory effects[20]. Through these discoveries, the concept of transcriptional kinetics as a determinant of riboswitch function has come into focus [2]. Transient structural forms such as anti-P1 exemplify a sophisticated mechanism of regulation at play, where the timing, duration and positioning of transcriptional pausing of RNAP relative to nascent RNA motifs is empowered to affect profound regulatory consequences. These emergent findings underscore the highly dynamic nature of RNA folding in the context of transcription and highlight the crucial roles of transient RNA structures in building the functional capacity of riboswitches to readily respond to cellular stimuli. This insight not only deepens our mechanistic understanding of RNA biology, but also enhances our broader understanding of the power of RNA-based gene regulation.

The Interconnection of Riboswitches and Cellular Components

The intricate, versatile and inherently dynamic landscape of RNA folding enables it to become significantly shaped not only by sequence elements but also by the dynamic interactions with other cellular constituents [21]. Riboswitches, which were traditionally viewed as autonomous molecular sensors, are part of a broader regulatory network that includes vital interactions with proteins such as RNAP [22] and transcription factors [23].

Understanding the multifaceted nature of riboswitches requires examining their influence on the behavior of associated transcription factors. Recent advances in fluorescent labeling and observation techniques have allowed for a detailed investigation into how the unique RNA folding patterns of riboswitches can modulate the kinetics of transcription factor interactions with RNAP[24,25]. A key discovery in this area involved tracking the binding kinetics of the transcription factor NusA [23,26]. NusA has an established genome-wide role in affecting transcription elongation and termination by modulating transcriptional pausing [27-29]. Its interactions with an elongation complex naturally paused downstream of a riboswitch were monitored to understand its influence on riboswitch function and vice versa. In these studies, it was observed that the specific transient structures formed by the riboswitch during transcription can significantly affect the binding kinetics of this versatile transcription factor. Therefore, the folding events and local structures within the RNA transcript can create a structural and kinetic landscape that either promotes or impairs the association and dissociation of NusA with the paused elongation complex (PEC). This interplay demonstrates that the riboswitch, through its conformational dynamics, has the ability to exert control over the activity of NusA and ultimately the rate of the macromolecular transcription machinery.

The interplay between RNA structures and transcription termination processes is markedly complex, particularly with regard to bacterial termination factor Rho. Rho-dependent termination requires a transcriptional pause, which is universal across all mechanisms of transcription termination, serving as a nexus point for the cessation of transcription (Figure 3) [30-32]. A recent single molecule study has offered insights into the mechanisms by which Rho operates [33]. The authors observed that the three known Rho-dependent termination routes can coexist within a single terminator system, each with distinctive dynamics influenced by transcriptional pausing. The most prevalent pathway identified in this study is where Rho initially binds to the nascent RNA and then catches up to the PEC (Figure 3A). The study also revealed that the efficiency of termination facilitated by a stand-by Rho, prebound to RNAP (Figure 3B), correlates positively with the length of transcriptional pauses as postulated in the allosteric model of Rho-mediated termination of transcription [31]. Significantly, the termination of certain riboswitches, such as mgtA and ribB [34,35], is largely governed by this delicate tuning of the stand-by Rho route, rather than the catch-up mechanism. This highlights the importance of understanding how riboswitch RNA structures modulate Rho-dependent termination. Riboswitches can influence transcriptional pausing and thus potentially control which Rho termination pathway is utilized, weaving an additional layer of regulation into the fabric of gene expression control.

Figure 3.

Riboswitches can potentially modulate Rho-mediated termination of transcription through two distinct mechanisms. (a) In the absence of ligand, the rut (Rho-Utilization) site is sequestered within an RNA structure, preventing Rho binding to the mRNA. In the presence of ligand, the rut site becomes accessible for Rho binding, which in turns hydrolyzes ATP to motor down the RNA and catch up with RNAP, promoting transcription termination. (b) In the allosteric model, Rho is always bound to the RNAP and is activated through ligand-mediated regulation of transcriptional pausing, providing a kinetic window for efficient termination of transcription.

Given this complex nature of transcription termination and the findings recently emerging from single-molecule assays, future research in riboswitch biology must consider the nuanced interplay between RNA structure, transcriptional pauses, RNAP dynamics, and the Rho termination factor. Understanding these interactions is essential for a comprehensive model of gene regulation, strengthening our grasp of how bacteria finely orchestrate the expression of their genome. These findings also challenge the conventional view of a passive riboswitch behavior by instead positioning these RNA elements as central, active drivers in the regulation of transcription processes.

The ability to monitor protein-RNA interactions in real-time by single molecule fluorescence tools in particular elucidates the synergistic nature of riboswitch regulation, replacing a simple ligand-sensor model wherein only the RNA structure adapts to affect RNA-based regulation of gene expression [21,36,37]. It underscores the complexity of regulatory mechanisms, where riboswitch activity is finely tuned through the confluence of multiple signals and interactions within the highly complex cellular environment. The appreciation of these riboswitch-protein interactions also opens up new avenues for interfacing with and manipulating the adaptive capabilities of cells to orchestrate precise gene expression responses to diverse environments.

Riboswitches Regulating Translation: Influence on Initiation and Transcription Coupling

In addition to controlling transcription outcomes, riboswitches – especially in Gram-negative bacteria – can play a crucial role in regulating translation, particularly at the earliest stage of translation initiation [38-41]. By adopting specific conformations upon ligand binding, riboswitches can control the accessibility of the ribosome binding site (RBS), directly or indirectly influencing whether translation of the downstream gene is allowed to start [42-44]. An exposed RBS allows ribosomes to bind and initiate protein synthesis, whereas a structurally sequestered RBS prevents translation, effectively silencing gene expression (Figure 1B).

The control over translation initiation by riboswitches is particularly important given the nature of transcription-translation coupling in especially Gram-negative bacteria [45]. This process is a hallmark of prokaryotic gene expression, where ribosomes initiate translation on nascent mRNA transcripts while they are still being synthesized by RNAP [46-48]. Such coupling takes advantage of the single-compartment architecture of the bacterial cell and is beneficial since it streamlines the process of gene expression, allowing for a coordinated and rapid response to changes within the cellular environment.

Given this overlap between transcription and translation right at the start of a transcript, where RNA structure matters most for bacterial gene expression[49], riboswitch-induced conformations that form co-transcriptionally are poised to have immediate effects on translation initiation [50]. Structured RNA elements can create physical barriers or alter the conformation of the mRNA in a way that directly influences RBS availability. This capability to modulate structural accessibility of the RBS gives riboswitches a powerful lever with which to fine-tune protein production in response to specific intracellular cues.

Moreover, since translation initiation occurs co-transcriptionally in bacteria, riboswitches that fold into inhibitory structures can do so in tandem with the transcription process. The outcome is a tightly regulated expression system where the fate of the mRNA — whether it is to be translated into protein or not — is determined in part by the folding dynamics of the riboswitch [50]. This ensures that gene expression is not just switched 'ON' or 'OFF’ but is precisely calibrated at the level of translation initiation, exemplifying the nuanced regulatory control exerted by riboswitches.

Transcription-translation coupling in bacteria serves multiple purposes, not only promoting efficient gene expression through reactivation of paused RNAP [51-54], but also protecting nascent mRNA from degradation. In the absence of ribosome binding and subsequent active translation elongation — conditions that can be orchestrated by riboswitch-mediated sequestration of the RBS — the unprotected mRNA may become vulnerable to degradation by nucleases (Figure 4) [55,56]. Furthermore, this uncoupled state may invite the intervention of the Rho termination factor, potentially inducing premature transcription termination through a process known as polarity (Figure 4) [57].

Figure 4.

Riboswitches at the nexus of gene expression regulation. RNAP pausing in the vicinity of the ribosome binding site and start codon allows proper coupling between the bacterial transcription and translation machineries. In the ligand-free state, ribosome binding promotes transcription reactivation of the RNAP paused state, leading to tight coupling between transcription and translation. Transcription factors such as NusA, NusG or S1 protein potentially allow fine-tuning of this process. The ligand-bound state inhibits translation initiation, leaving the RNAP and the mRNA unprotected from safeguard mechanisms. Therefore, transcription can be prematurely terminated through the action of Rho protein (in a process termed polarity). Spurious transcripts that escape termination are exposed to degradation by cellular ribonucleases.

This reliance on the coupling between transcription and translation for successful gene expression underscores the potential of riboswitches to exert control and accurately modulate the activity of the entire cellular machinery. By governing when to expose or hide the RBS, riboswitches not only determine the fate of translation initiation but also influence the overall stability and life cycle of the mRNA transcript. Polarity, as such, may represent a key regulatory mechanism utilized by most translationally acting riboswitches, ensuring that gene expression is tightly regulated across multiple layers and safeguarded against unwarranted protein production. The interplay between RNA structure, ribosome action, and the susceptibility of mRNA to degradation and termination highlights the sophistication of bacterial adaptability and the critical role riboswitches play in orchestrating these complex biological processes.

Future Directions in Riboswitch Research: Structural Insights and Integrative Approaches

The future of riboswitch research holds immense potential for improving our understanding of RNA-based regulation of gene expression at multiple levels, with the prospect of informing intervention tools. One of the critical needs is the identification of transcriptional intermediates or transient structures that play pivotal roles during the co-transcriptional folding process [58]. Pinpointing these transient states will lead to a more nuanced understanding of how riboswitches kinetically partition between different functional conformations. Advanced imaging techniques such as single particle cryogenic electron microscopy (cryo-EM) and in situ cryogenic electron tomography (cryo-ET), traditionally known for their high-resolution structural revelations, appear to be on the verge of major discoveries regarding dynamic RNA and RNA-protein complexes, potentially providing snapshots of riboswitches [59,60] in action and revealing the dynamic processes that underlie their regulatory functions in complex with the cellular network [61-65].

The advent of time-resolved cryo-EM techniques also has the potential to revolutionize our understanding of the kinetic aspects of riboswitch function, as it merges the capability for structural elucidation with temporal resolution [66,67]. By capturing multiple conformational states along a reaction pathway, time-resolved cryo-EM could answer longstanding questions about the folding kinetics and ligand response of riboswitches, situating this method at the forefront of exploring the dynamic RNA world.

In light of the dozens of known, often combinatorically acting bacterial transcription factors[68], a comprehensive survey of riboswitch interactions with a broader range of cellular components remains a significant endeavor. These components further include the Rho termination factor, which enforces the timely termination of transcription, ribonucleases like RNase E [55,69,70], crucial for RNA processing and degradation, or protein chaperons such as Hfq [71,72] that can potentially alter the specific riboswitch architecture. Understanding how riboswitches interact with transcription factors, the translation machinery, and small RNAs (sRNAs) regulating in trans will further illuminate their integrative role within the cellular regulatory network. By including time-resolved studies with these various factors at the single molecule level, researchers will build a more integrated picture of how riboswitches modulate not just their target mRNA but also how they influence and are influenced by the wider molecular symphony within the cell.

Expanding in vivo methodologies represents another frontier in riboswitch research. Techniques such as single particle tracking [73], and in vivo RNA structure probing [74-77] can disclose the behavior and regulatory impact of riboswitches in their native cellular context. These methods can provide real-time insights into the stochastic nature of riboswitch-regulated pathways and contribute vital information on the cellular heterogeneity of regulatory outcomes.

As research continues to advance, these diverse approaches will shape a comprehensive and integrative view of riboswitches. Concerted efforts across structural biology, molecular genetics, and cellular biology promises to unravel the sophisticated strategies that bacteria employ to regulate genes through RNA elements, contributing to science's broader understanding of life’s molecular orchestration and how to interface with it.

Data availability

No data were used for the research described in the article.