The results establish ρ analysis as a general framework to probe RNA conformational pathways and function and uncover multiple folding pathways and modulation in pathway flux that are readily understood from the energetic properties of the constituent RNA motifs.
Abstract
The modular structure and energetics of RNA simplifies its folding. Leveraging this modularity, we introduce Rho (ρ) analysis to systematically dissect RNA conformational pathways. ρ analysis uses isolated RNA secondary or tertiary contacts as external standards to provide insights not possible via the “internal” comparisons of traditional ϕ analysis. Equivalent effects of a mutation on the folding rate constant of the RNA of interest and the thermodynamic stability of the isolated contact indicate that the mutated interaction is fully formed prior to the rate-limiting transition state; the absence of a kinetic effect indicates that the interaction is formed after this transition state. Comparisons with properties of the isolated contact provide additional insights about conformational pathways. We demonstrate ρ analysis by dissecting Tetrahymena group I intron folding pathways, using a split intron in which the P5abc subdomain assembles with the intron core through three tertiary contacts. We uncover multiple folding pathways and modulation in pathway flux that are readily understood from the energetic properties of the constituent RNA motifs. Extending these concepts to RNA-guided DNA recognition by CRISPR-Cas12a, crRNA–DNA mismatches give substantial ϕ values across much of the target, indicating a late transition state in binding. Thermodynamic penalties from mismatches support modular base-pairing energetics and define an upper bound on DNA target specificity. Our results establish ρ analysis as a general framework to probe RNA conformational pathways and function. It is straightforward to implement and can be readily applied in vitro and in cells.
It is concluded that the early emergence of the Rossmann fold reflects the chemical and physical constraints of protein folding, explaining both its profound antiquity and sustained longevity.
Koh Seya, Tatsuya Corlett, Hamza Giaffar et al.· bioRxiv· 0 citations
RNA interactions are a key contributor to the formation and disassembly of intracellular protein condensates. Although some proteins utilize specific RNA-binding domains, these processes can also be mediated by charge interactions with intrinsically disordered regions. Due to the dynamic nature of these systems, investigating the underlying specificity and stoichiometry remains challenging. Here, we demonstrate that single-molecule mass measurements with mass photometry can capture RNA-protein interactions in phase-separated protein systems. Using the approach to investigate RNA-mediated phase shifts of tau condensates, we find that increasing the RNA concentration, which promotes phase re-entry, results in RNA-mediated tau multimerization, where each tau monomer binds a linear RNA sequence of approximately 30 nucleotides. Solution NMR and native mass spectrometry confirm the formation of stable complexes between RNA and the basic proline-rich and repeat domains of tau, which have a net charge of -29. Our findings demonstrate that mass photometry can distinguish between charge neutralization, which drives coacervation, and complex formation, which mediates phase re-entry, making it a highly complementary tool for the study of RNA-mediated phase separation.
Axel Leppert, Jesper Shiapan, Irena Papageorgiou et al.· bioRxiv· 0 citations
A mechanistic model of de novo folding initiation during biosynthesis is infer and a complete atomistic description of a co-translational folding pathway is provided by linking the folding nucleus to downstream partially structured intermediates and the native state is provided.
Ivana V. Bukvin, Julian O. Streit, Tomasz Włodarski et al.· bioRxiv· 0 citations
It is shown that short nucleic acids containing Gquadruplex (G4) structure can also catalyze protein folding and uncovers a previously underappreciated role for nucleic acid in proteostasis and offers a new strategy for studying nucleic acid structure-function relationship at residue level.
RNA molecules populate complex structural landscapes that are continuously reshaped throughout the RNA lifecycle by equilibrium and non-equilibrium processes. Resolving these structural landscapes represents a central challenge in RNA biochemistry. We review recent advances in RNA chemical probing, sequencing, and computational deconvolution technologies that are revolutionizing our ability to measure the complexities of RNA folding in cells and the deep involvement of these complexities in RNA functional mechanisms. We highlight new methods for deconvolving structural ensembles, distinguishing isoform-specific architectures using long-read sequencing, capturing co-transcriptional folding intermediates in vivo, and measuring higher-order RNA structures while also underscoring remaining challenges. We conclude by outlining future directions in probe development, sequencing, and integrative modeling, and discuss how resolving RNA structural ensembles with increasingly high resolution will likely reveal new therapeutic opportunities to selectively target functional RNA heterogeneity.
Anthony M. Mustoe, Danny Incarnato· Current Opinion in Structura...· 0 citations
RNA polymerase II (RNAPII) drives gene expression through iterative nucleotide addition cycles (NACs) comprising translocation, substrate binding, and catalysis. The lack of pre-catalysis and post-catalysis intermediates has precluded a complete mechanistic understanding of the NAC. Here we present 31 Cryo-electron Microscopy structures (with 43 maps) capturing distinct stages of Saccharomyces cerevisiae RNAPII elongation complex (EC) NAC, including previously intractable transition intermediates. We establish a continuous spectrum of RNAPII EC structural dynamics during the NAC, which can be divided into two coordinated phases: a substrate-induced EC tightening phase and a post-catalysis EC relaxation phase. For the substrate-induced EC tightening phase, the substrate binding initiates allosteric conformational changes across the entire RNAPII EC, including Trigger Loop folding, funnel closure, clamp closure, transcription bubble ordering, and precise alignment of the RNA 3′-end with substrate to form a catalysis-competent configuration. For the post-catalysis EC relaxation phase, we capture the long-sought, short-lived post-catalysis product state and identify a series of intermediates that reveal a reverse conformational transition that facilitates rapid translocation. Together, our findings define a comprehensive structural and dynamic framework for RNAPII NAC, yielding a “molecular movie” of RNAPII in action and revealing a fundamental principle by which the enzyme balances speed and fidelity through coordinated conformational dynamics. RNA polymerase II catalyses transcription through nucleotide addition cycles, but key structural intermediates were unresolved. Here, the authors present cryo‑EM snapshots capturing the elongation cycle, revealing coordinated conformational dynamics that balance catalytic efficiency and fidelity.
Gangshun Yi, Qingrong Li, H. Holmberg et al.· Nature Communications· 0 citations