Ions surround nucleic acids in what is referred to as an ion atmosphere. As a result, the folding and dynamics of RNA and DNA and their complexes with proteins and with each other cannot be ...understood without a reasonably sophisticated appreciation of these ions' electrostatic interactions. However, the underlying behavior of the ion atmosphere follows physical rules that are distinct from the rules of site binding that biochemists are most familiar and comfortable with. The main goal of this review is to familiarize nucleic acid experimentalists with the physical concepts that underlie nucleic acid-ion interactions. Throughout, we provide practical strategies for interpreting and analyzing nucleic acid experiments that avoid pitfalls from oversimplified or incorrect models. We briefly review the status of theories that predict or simulate nucleic acid-ion interactions and experiments that test these theories. Finally, we describe opportunities for going beyond phenomenological fits to a next-generation, truly predictive understanding of nucleic acid-ion interactions.
Quantitative measurements of biomolecule associations are central to biological understanding and are needed to build and test predictive and mechanistic models. Given the advances in high-throughput ...technologies and the projected increase in the availability of binding data, we found it especially timely to evaluate the current standards for performing and reporting binding measurements. A review of 100 studies revealed that in most cases essential controls for establishing the appropriate incubation time and concentration regime were not documented, making it impossible to determine measurement reliability. Moreover, several reported affinities could be concluded to be incorrect, thereby impacting biological interpretations. Given these challenges, we provide a framework for a broad range of researchers to evaluate, teach about, perform, and clearly document high-quality equilibrium binding measurements. We apply this framework and explain underlying fundamental concepts through experimental examples with the RNA-binding protein Puf4.
RNAs fold into 3D structures that range from simple helical elements to complex tertiary structures and quaternary ribonucleoprotein assemblies. The functions of many regulatory RNAs depend on how ...their 3D structure changes in response to a diverse array of cellular conditions. In this Review, we examine how the structural characterization of RNA as dynamic ensembles of conformations, which form with different probabilities and at different timescales, is improving our understanding of RNA function in cells. We discuss the mechanisms of gene regulation by microRNAs, riboswitches, ribozymes, post-transcriptional RNA modifications and RNA-binding proteins, and how the cellular environment and processes such as liquid-liquid phase separation may affect RNA folding and activity. The emerging RNA-ensemble-function paradigm is changing our perspective and understanding of RNA regulation, from in vitro to in vivo and from descriptive to predictive.
Phosphoryl-transfer reactions are central to biology. These reactions also have some of the slowest nonenzymatic rates and thus require enormous rate accelerations from biological catalysts. Despite ...the central importance of phosphoryl transfer and the fascinating catalytic challenges it presents, substantial confusion persists about the properties of these reactions. This confusion exists despite decades of research on the chemical mechanisms underlying these reactions. Here we review phosphoryl-transfer reactions with the goal of providing the reader with the conceptual and experimental background to understand this body of work, to evaluate new results and proposals, and to apply this understanding to enzymes. We describe likely resolutions to some controversies, while emphasizing the limits of our current approaches and understanding. We apply this understanding to enzyme-catalyzed phosphoryl transfer and provide illustrative examples of how this mechanistic background can guide and deepen our understanding of enzymes and their mechanisms of action. Finally, we present important future challenges for this field.
Electrostatics are central to all aspects of nucleic acid behavior, including their folding, condensation, and binding to other molecules, and the energetics of these processes are profoundly ...influenced by the ion atmosphere that surrounds nucleic acids. Given the highly complex and dynamic nature of the ion atmosphere, understanding its properties and effects will require synergy between computational modeling and experiment. Prior computational models and experiments suggest that cation occupancy in the ion atmosphere depends on the size of the cation. However, the computational models have not been independently tested, and the experimentally observed effects were small. Here, we evaluate a computational model of ion size effects by experimentally testing a blind prediction made from that model, and we present additional experimental results that extend our understanding of the ion atmosphere. Giambasu et al. developed and implemented a three-dimensional reference interaction site (3D-RISM) model for monovalent cations surrounding DNA and RNA helices, and this model predicts that Na+ would outcompete Cs+ by 1.8–2.1-fold; i.e., with Cs+ in 2-fold excess of Na+ the ion atmosphere would contain an equal number of each cation (Nucleic Acids Res. 2015, 43, 8405). However, our ion counting experiments indicate that there is no significant preference for Na+ over Cs+. There is an ∼25% preferential occupancy of Li+ over larger cations in the ion atmosphere but, counter to general expectations from existing models, no size dependence for the other alkali metal ions. Further, we followed the folding of the P4–P6 RNA and showed that differences in folding with different alkali metal ions observed at high concentration arise from cation–anion interactions and not cation size effects. Overall, our results provide a critical test of a computational prediction, fundamental information about ion atmosphere properties, and parameters that will aid in the development of next-generation nucleic acid computational models.
The Story of RNA Folding, as Told in Epochs Herschlag, Daniel; Bonilla, Steve; Bisaria, Namita
Cold Spring Harbor perspectives in biology,
10/2018, Letnik:
10, Številka:
10
Journal Article
Recenzirano
Odprti dostop
The past decades have witnessed tremendous developments in our understanding of RNA biology. At the core of these advances have been studies aimed at discerning RNA structure and at understanding the ...forces that influence the RNA folding process. It is easy to take the present state of understanding for granted, but there is much to be learned by considering the path to our current understanding, which has been tortuous, with the birth and death of models, the adaptation of experimental tools originally developed for characterization of protein structure and catalysis, and the development of novel tools for probing RNA. In this review we tour the stages of RNA folding studies, considering them as "epochs" that can be generalized across scientific disciplines. These epochs span from the discovery of catalytic RNA, through biophysical insights into the putative primordial RNA World, to characterization of structured RNAs, the building and testing of models, and, finally, to the development of models with the potential to yield generalizable predictive and quantitative models for RNA conformational, thermodynamic, and kinetic behavior. We hope that this accounting will aid others as they navigate the many fascinating questions about RNA and its roles in biology, in the past, present, and future.
RNA-binding proteins (RBPs) have roles in the regulation of many post-transcriptional steps in gene expression, but relatively few RBPs have been systematically studied. We searched for the RNA ...targets of 40 proteins in the yeast Saccharomyces cerevisiae: a selective sample of the approximately 600 annotated and predicted RBPs, as well as several proteins not annotated as RBPs. At least 33 of these 40 proteins, including three of the four proteins that were not previously known or predicted to be RBPs, were reproducibly associated with specific sets of a few to several hundred RNAs. Remarkably, many of the RBPs we studied bound mRNAs whose protein products share identifiable functional or cytotopic features. We identified specific sequences or predicted structures significantly enriched in target mRNAs of 16 RBPs. These potential RNA-recognition elements were diverse in sequence, structure, and location: some were found predominantly in 3'-untranslated regions, others in 5'-untranslated regions, some in coding sequences, and many in two or more of these features. Although this study only examined a small fraction of the universe of yeast RBPs, 70% of the mRNA transcriptome had significant associations with at least one of these RBPs, and on average, each distinct yeast mRNA interacted with three of the RBPs, suggesting the potential for a rich, multidimensional network of regulation. These results strongly suggest that combinatorial binding of RBPs to specific recognition elements in mRNAs is a pervasive mechanism for multi-dimensional regulation of their post-transcriptional fate.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Biomolecules form dynamic ensembles of many inter-converting conformations which are key for understanding how they fold and function. However, determining ensembles is challenging because the ...information required to specify atomic structures for thousands of conformations far exceeds that of experimental measurements. We addressed this data gap and dramatically simplified and accelerated RNA ensemble determination by using structure prediction tools that leverage the growing database of RNA structures to generate a conformation library. Refinement of this library with NMR residual dipolar couplings provided an atomistic ensemble model for HIV-1 TAR, and the model accuracy was independently supported by comparisons to quantum-mechanical calculations of NMR chemical shifts, comparison to a crystal structure of a substate, and through designed ensemble redistribution via atomic mutagenesis. Applications to TAR bulge variants and more complex tertiary RNAs support the generality of this approach and the potential to make the determination of atomic-resolution RNA ensembles routine.
Intermediates play important roles in RNA folding but can be difficult to characterize when short-lived or not significantly populated. By combining (15)N relaxation dispersion NMR with chemical ...probing, we visualized a fast (kex=k1+k-1≈423 s(-1)) secondary structural switch directed towards a low-populated (∼3%) partially folded intermediate in tertiary folding of the P5abc subdomain of the 'Tetrahymena' group I intron ribozyme. The secondary structure switch changes the base-pairing register across the P5c hairpin, creating a native-like structure, and occurs at rates of more than two orders of magnitude faster than tertiary folding. The switch occurs robustly in the absence of tertiary interactions, Mg(2+) or even when the hairpin is excised from the three-way junction. Fast, highly modular secondary structural switches may be quite common during RNA tertiary folding where they may help smoothen the folding landscape by allowing folding to proceed efficiently via additional pathways.
Reprogramming of a gene's expression pattern by acquisition and loss of sequences recognized by specific regulatory RNA binding proteins may be a major mechanism in the evolution of biological ...regulatory programs. We identified that RNA targets of Puf3 orthologs have been conserved over 100-500 million years of evolution in five eukaryotic lineages. Focusing on Puf proteins and their targets across 80 fungi, we constructed a parsimonious model for their evolutionary history. This model entails extensive and coordinated changes in the Puf targets as well as changes in the number of Puf genes and alterations of RNA binding specificity including that: 1) Binding of Puf3 to more than 200 RNAs whose protein products are predominantly involved in the production and organization of mitochondrial complexes predates the origin of budding yeasts and filamentous fungi and was maintained for 500 million years, throughout the evolution of budding yeast. 2) In filamentous fungi, remarkably, more than 150 of the ancestral Puf3 targets were gained by Puf4, with one lineage maintaining both Puf3 and Puf4 as regulators and a sister lineage losing Puf3 as a regulator of these RNAs. The decrease in gene expression of these mRNAs upon deletion of Puf4 in filamentous fungi (N. crassa) in contrast to the increase upon Puf3 deletion in budding yeast (S. cerevisiae) suggests that the output of the RNA regulatory network is different with Puf4 in filamentous fungi than with Puf3 in budding yeast. 3) The coregulated Puf4 target set in filamentous fungi expanded to include mitochondrial genes involved in the tricarboxylic acid (TCA) cycle and other nuclear-encoded RNAs with mitochondrial function not bound by Puf3 in budding yeast, observations that provide additional evidence for substantial rewiring of post-transcriptional regulation. 4) Puf3 also expanded and diversified its targets in filamentous fungi, gaining interactions with the mRNAs encoding the mitochondrial electron transport chain (ETC) complex I as well as hundreds of other mRNAs with nonmitochondrial functions. The many concerted and conserved changes in the RNA targets of Puf proteins strongly support an extensive role of RNA binding proteins in coordinating gene expression, as originally proposed by Keene. Rewiring of Puf-coordinated mRNA targets and transcriptional control of the same genes occurred at different points in evolution, suggesting that there have been distinct adaptations via RNA binding proteins and transcription factors. The changes in Puf targets and in the Puf proteins indicate an integral involvement of RNA binding proteins and their RNA targets in the adaptation, reprogramming, and function of gene expression.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
