The human genome project's lasting legacies are the emerging insights into human physiology and disease, and the ascendance of biology as the dominant science of the 21st century. Sequencing revealed ...that >90% of the human genome is not coding for proteins, as originally thought, but rather is overwhelmingly transcribed into non-protein coding, or non-coding, RNAs (ncRNAs). This discovery initially led to the hypothesis that most genomic DNA is "junk", a term still championed by some geneticists and evolutionary biologists. In contrast, molecular biologists and biochemists studying the vast number of transcripts produced from most of this genome "junk" often surmise that these ncRNAs have biological significance. What gives? This essay contrasts the two opposing, extant viewpoints, aiming to explain their bases, which arise from distinct reference frames of the underlying scientific disciplines. Finally, it aims to reconcile these divergent mindsets in hopes of stimulating synergy between scientific fields.
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.
Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and ...stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.
Commensal and pathogenic bacteria continuously evolve to survive in diverse ecological niches by efficiently coordinating gene expression levels in their ever-changing environments. Regulation ...through the RNA transcript itself offers a faster and more cost-effective way to adapt than protein-based mechanisms and can be leveraged for diagnostic or antimicrobial purposes. However, RNA can fold into numerous intricate, not always functional structures that both expand and obscure the plethora of roles that regulatory RNAs serve within the cell. Here, we review the current knowledge of bacterial non-coding RNAs in relation to their folding pathways and interactions. We posit that co-transcriptional folding of these transcripts ultimately dictates their downstream functions. Elucidating the spatiotemporal folding of non-coding RNAs during transcription therefore provides invaluable insights into bacterial pathogeneses and predictive disease diagnostics. Finally, we discuss the implications of co-transcriptional folding andapplications of RNAs for therapeutics and drug targets.
Bacterial riboswitches couple small-molecule ligand binding to RNA conformational changes that widely regulate gene expression, rendering them potential targets for antibiotic intervention. Despite ...structural insights, the ligand-mediated folding mechanisms of riboswitches are still poorly understood. Using single-molecule fluorescence resonance energy transfer (smFRET), we have investigated the folding mechanism of an H-type pseudoknotted preQ1 riboswitch in dependence of Mg2+ and three ligands of distinct affinities. We show that, in the absence of Mg2+, both weakly and strongly bound ligands promote pseudoknot docking through an induced-fit mechanism. By contrast, addition of as low as 10 μM Mg2+ generally shifts docking toward conformational selection by stabilizing a folded-like conformation prior to ligand binding. Supporting evidence from transition-state analysis further highlights the particular importance of stacking interactions during induced-fit and of specific hydrogen bonds during conformational selection. Our mechanistic dissection provides unprecedented insights into the intricate synergy between ligand- and Mg2+-mediated RNA folding.
Biomolecular condensates are membraneless cellular compartments generated by phase separation that regulate a broad variety of cellular functions by enriching some biomolecules while excluding ...others. Live-cell single particle tracking of individual fluorophore-labeled condensate components has provided insights into a condensate’s mesoscopic organization and biological functions, such as revealing the recruitment, translation, and decay of RNAs within ribonucleoprotein (RNP) granules. Specifically, during dual-color tracking, one imaging channel provides a time series of individual biomolecule locations, while the other channel monitors the location of the condensate relative to these molecules. Therefore, an accurate assessment of a condensate’s boundary is critical for combined live-cell single particle-condensate tracking. Despite its importance, a quantitative benchmarking and objective comparison of the various available boundary detection methods is missing due to the lack of an absolute ground truth for condensate images. Here, we use synthetic data of defined ground truth to generate noise-overlaid images of condensates with realistic phase separation parameters to benchmark the most commonly used methods for condensate boundary detection, including an emerging machine-learning method. We find that it is critical to carefully choose an optimal boundary detection method for a given dataset to obtain accurate measurements of single particle-condensate interactions. The criteria proposed in this study to guide the selection of an optimal boundary detection method can be broadly applied to imaging-based studies of condensates.
Biology arises from the crowded molecular environment of the cell, rendering it a challenge to understand biological pathways based on the reductionist, low‐concentration in vitro conditions ...generally employed for mechanistic studies. Recent evidence suggests that low‐affinity interactions between cellular biopolymers abound, with still poorly defined effects on the complex interaction networks that lead to the emergent properties and plasticity of life. Mass‐action considerations are used here to underscore that the sheer number of weak interactions expected from the complex mixture of cellular components significantly shapes biological pathway specificity. In particular, on‐pathway—i.e., “functional”—become those interactions thermodynamically and kinetically stable enough to survive the incessant onslaught of the many off‐pathway (“nonfunctional”) interactions. Consequently, to better understand the molecular biology of the cell a further paradigm shift is needed toward mechanistic experimental and computational approaches that probe intracellular diversity and complexity more directly. Also see the video here https://youtu.be/T19X_zYaBzg.
How exactly specific biological pathways and eventually life arise from the crowded molecular environment of the cell is a problem that will require a paradigm shift toward mechanistic experimental and computational approaches that probe intracellular diversity and complexity more directly.
During pre-mRNA splicing, a central step in the expression and regulation of eukaryotic genes, the spliceosome selects splice sites for intron excision and exon ligation. In doing so, the spliceosome ...must distinguish optimal from suboptimal splice sites. At the catalytic stage of splicing, suboptimal splice sites are repressed by the DEAH-box ATPases Prp16 and Prp22. Here, using budding yeast, we show that these ATPases function further by enabling the spliceosome to search for and utilize alternative branch sites and 3′ splice sites. The ATPases facilitate this search by remodeling the splicing substrate to disengage candidate splice sites. Our data support a mechanism involving 3′ to 5′ translocation of the ATPases along substrate RNA and toward a candidate site, but, surprisingly, not across the site. Thus, our data implicate DEAH-box ATPases in acting at a distance by pulling substrate RNA from the catalytic core of the spliceosome.
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•The DEAH-box ATPase Prp16 enables alternative branch site selection•The DEAH-box ATPase Prp22 promotes alternative 3′ splice site selection•Prp16 and Prp22 both repress suboptimal sites by disengaging the splice sites•Prp16 and Prp22 remodel the substrate without translocating through their targets
Two DEAH-box ATPases enable the spliceosome to search for and utilize alternative branch sites and 3′ splice sites by disengaging suboptimal sites from the splicing machinery through a mechanism that suggests action at a distance by RNA pulling.
Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is ...covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.
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