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Open Biology Aug 2016The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for... (Review)
Review
The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for producing recombinant proteins. In this post-genomic era, challenges remain to rapidly express and purify large numbers of proteins for academic and commercial purposes in a high-throughput manner. In this review, we describe several state-of-the-art approaches that are suitable for the cloning, expression and purification, conducted in parallel, of numerous molecules, and we discuss recent progress related to soluble protein expression, mRNA folding, fusion tags, post-translational modification and production of membrane proteins. Moreover, we address the ongoing efforts to overcome various challenges faced in protein expression in E. coli, which could lead to an improvement of the current system from trial and error to a predictable and rational design.
Topics: Cloning, Molecular; Escherichia coli; Gene Expression; Protein Engineering; Protein Processing, Post-Translational; RNA Folding; RNA, Messenger; Recombinant Proteins
PubMed: 27581654
DOI: 10.1098/rsob.160196 -
Journal of Molecular Biology Sep 2022Recent advances in interrogating RNA folding dynamics have shown the classical model of RNA folding to be incomplete. Here, we pose three prominent questions for the... (Review)
Review
Recent advances in interrogating RNA folding dynamics have shown the classical model of RNA folding to be incomplete. Here, we pose three prominent questions for the field that are at the forefront of our understanding of the importance of RNA folding dynamics for RNA function. The first centers on the most appropriate biophysical framework to describe changes to the RNA folding energy landscape that a growing RNA chain encounters during transcriptional elongation. The second focuses on the potential ubiquity of strand displacement - a process by which RNA can rapidly change conformations - and how this process may be generally present in broad classes of seemingly different RNAs. The third raises questions about the potential importance and roles of cellular protein factors in RNA conformational switching. Answers to these questions will greatly improve our fundamental knowledge of RNA folding and function, drive biotechnological advances that utilize engineered RNAs, and potentially point to new areas of biology yet to be discovered.
Topics: Kinetics; RNA; RNA Folding
PubMed: 35659535
DOI: 10.1016/j.jmb.2022.167665 -
Journal of Molecular Biology Sep 2022RNA folding free energy change parameters are widely used to predict RNA secondary structure and to design RNA sequences. These parameters include terms for the folding...
RNA folding free energy change parameters are widely used to predict RNA secondary structure and to design RNA sequences. These parameters include terms for the folding free energies of helices and loops. Although the full set of parameters has only been traditionally available for the four common bases and backbone, it is well known that covalent modifications of nucleotides are widespread in natural RNAs. Covalent modifications are also widely used in engineered sequences. We recently derived a full set of nearest neighbor terms for RNA that includes N-methyladenosine (mA). In this work, we test the model using 98 optical melting experiments, matching duplexes with or without N-methylation of A. Most experiments place RRACH, the consensus site of N-methylation, in a variety of contexts, including helices, bulge loops, internal loops, dangling ends, and terminal mismatches. For matched sets of experiments that include either A or mA in the same context, we find that the parameters for mA are as accurate as those for A. Across all experiments, the root mean squared deviation between estimated and experimental free energy changes is 0.67 kcal/mol. We used the new experimental data to refine the set of nearest neighbor parameter terms for mA. These parameters enable prediction of RNA secondary structures including mA, which can be used to model how N-methylation of A affects RNA structure.
Topics: Adenosine; Entropy; RNA; RNA Folding
PubMed: 35588868
DOI: 10.1016/j.jmb.2022.167632 -
Viruses Oct 2020Viral RNA genomes change shape as virus particles disassemble, form replication complexes, attach to ribosomes for translation, evade host defense mechanisms, and... (Review)
Review
Viral RNA genomes change shape as virus particles disassemble, form replication complexes, attach to ribosomes for translation, evade host defense mechanisms, and assemble new virus particles. These structurally dynamic RNA shapeshifters present a challenging RNA folding problem, because the RNA sequence adopts multiple structures and may sometimes contain regions of partial disorder. Recent advances in high resolution asymmetric cryoelectron microscopy and chemical probing provide new ways to probe the degree of structure and disorder, and have identified more than one conformation in dynamic equilibrium in viral RNA. Chemical probing and the Detection of RNA Folding Ensembles using Expectation Maximization (DREEM) algorithm has been applied to studies of the dynamic equilibrium conformations in HIV RNA in vitro, in virio, and in vivo. This new type of data provides insight into important questions about virus assembly mechanisms and the fundamental physical forces driving virus particle assembly.
Topics: Base Sequence; Cryoelectron Microscopy; Genome, Viral; Nucleic Acid Conformation; RNA Folding; RNA, Viral; Virion; Virus Assembly
PubMed: 33027988
DOI: 10.3390/v12101126 -
Journal of Biotechnology Nov 2017In the realm of nucleic acid structures, secondary structure forms a conceptually important intermediate level of description and explains the dominating part of the... (Review)
Review
In the realm of nucleic acid structures, secondary structure forms a conceptually important intermediate level of description and explains the dominating part of the free energy of structure formation. Secondary structures are well conserved over evolutionary time-scales and for many classes of RNAs evolve slower than the underlying primary sequences. Given the close link between structure and function, secondary structure is routinely used as a basis to explain experimental findings. Recent technological advances, finally, have made it possible to assay secondary structure directly using high throughput methods. From a computational biology point of view, secondary structures have a special role because they can be computed efficiently using exact dynamic programming algorithms. In this contribution we provide a short overview of RNA folding algorithms, recent additions and variations and address methods to align, compare, and cluster RNA structures, followed by a tabular summary of the most important software suites in the fields.
Topics: Algorithms; Computational Biology; Nucleic Acid Conformation; RNA; RNA Folding; Sequence Analysis, RNA; Software
PubMed: 28690134
DOI: 10.1016/j.jbiotec.2017.07.007 -
Current Opinion in Structural Biology Dec 2019RNA structure underpins many essential functions in biology. New chemical reagents and techniques for probing RNA structure in living cells have emerged in recent years.... (Review)
Review
RNA structure underpins many essential functions in biology. New chemical reagents and techniques for probing RNA structure in living cells have emerged in recent years. High-throughput, genome-wide techniques such as Structure-seq2 and DMS-MaPseq exploit nucleobase modification by dimethylsulfate (DMS) to obtain complete structuromes, and are applicable to multiple domains of life and conditions. New reagents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), glyoxal, and nicotinoyl azide (NAz) greatly expand the capabilities of nucleobase probing in cells. Additionally, ribose-targeting reagents in selective 2'-hydroxyl acylation and primer extension (SHAPE) detect RNA flexibility in vivo. These techniques, coupled with crosslinking nucleobases in psoralen analysis of RNA interactions and structures (PARIS), provide new and diverse ways to elucidate RNA secondary and tertiary structure in vivo and genome-wide.
Topics: Models, Molecular; Molecular Structure; Nucleic Acid Conformation; RNA; RNA Folding; Sulfuric Acid Esters
PubMed: 31521910
DOI: 10.1016/j.sbi.2019.07.008 -
Science (New York, N.Y.) Sep 2021The human small subunit processome mediates early maturation of the small ribosomal subunit by coupling RNA folding to subsequent RNA cleavage and processing steps. We...
The human small subunit processome mediates early maturation of the small ribosomal subunit by coupling RNA folding to subsequent RNA cleavage and processing steps. We report the high-resolution cryo–electron microscopy structures of maturing human small subunit (SSU) processomes at resolutions of 2.7 to 3.9 angstroms. These structures reveal the molecular mechanisms that enable crucial progressions during SSU processome maturation. RNA folding states within these particles are communicated to and coordinated with key enzymes that drive irreversible steps such as targeted exosome-mediated RNA degradation, protein-guided site-specific endonucleolytic RNA cleavage, and tightly controlled RNA unwinding. These conserved mechanisms highlight the SSU processome’s impressive structural plasticity, which endows this 4.5-megadalton nucleolar assembly with the distinctive ability to mature the small ribosomal subunit from within.
Topics: Cell Nucleolus; Cryoelectron Microscopy; DEAD-box RNA Helicases; Humans; RNA Cleavage; RNA Folding; RNA Splicing Factors; RNA Stability; RNA, Small Nucleolar
PubMed: 34516797
DOI: 10.1126/science.abj5338 -
PLoS Computational Biology Apr 2022The 3-dimensional fold of an RNA molecule is largely determined by patterns of intramolecular hydrogen bonds between bases. Predicting the base pairing network from the...
The 3-dimensional fold of an RNA molecule is largely determined by patterns of intramolecular hydrogen bonds between bases. Predicting the base pairing network from the sequence, also referred to as RNA secondary structure prediction or RNA folding, is a nondeterministic polynomial-time (NP)-complete computational problem. The structure of the molecule is strongly predictive of its functions and biochemical properties, and therefore the ability to accurately predict the structure is a crucial tool for biochemists. Many methods have been proposed to efficiently sample possible secondary structure patterns. Classic approaches employ dynamic programming, and recent studies have explored approaches inspired by evolutionary and machine learning algorithms. This work demonstrates leveraging quantum computing hardware to predict the secondary structure of RNA. A Hamiltonian written in the form of a Binary Quadratic Model (BQM) is derived to drive the system toward maximizing the number of consecutive base pairs while jointly maximizing the average length of the stems. A Quantum Annealer (QA) is compared to a Replica Exchange Monte Carlo (REMC) algorithm programmed with the same objective function, with the QA being shown to be highly competitive at rapidly identifying low energy solutions. The method proposed in this study was compared to three algorithms from literature and, despite its simplicity, was found to be competitive on a test set containing known structures with pseudoknots.
Topics: Algorithms; Computational Biology; Computers; Computing Methodologies; Nucleic Acid Conformation; Quantum Theory; RNA; RNA Folding
PubMed: 35404931
DOI: 10.1371/journal.pcbi.1010032 -
Methods in Enzymology 2019Riboswitches are RNA elements that recognize diverse chemical and biomolecular inputs, and transduce this recognition process to genetic, fluorescent, and other...
Riboswitches are RNA elements that recognize diverse chemical and biomolecular inputs, and transduce this recognition process to genetic, fluorescent, and other engineered outputs using RNA conformational changes. These systems are pervasive in cellular biology and are a promising biotechnology with applications in genetic regulation and biosensing. Here, we derive a simple expression bounding the activation ratio-the proportion of RNA in the active vs. inactive states-for both ON and OFF riboswitches that operate near thermodynamic equilibrium: 1+[I]/K, where [I] is the input ligand concentration and K is the intrinsic dissociation constant of the aptamer module toward the input ligand. A survey of published studies of natural and synthetic riboswitches confirms that the vast majority of empirically measured activation ratios have remained well below this thermodynamic limit. A few natural and synthetic riboswitches achieve activation ratios close to the limit, and these molecules highlight important principles for achieving high riboswitch performance. For several applications, including "light-up" fluorescent sensors and chemically-controlled CRISPR/Cas complexes, the thermodynamic limit has not yet been achieved, suggesting that current tools are operating at suboptimal efficiencies. Future riboswitch studies will benefit from comparing observed activation ratios to this simple expression for the optimal activation ratio. We present experimental and computational suggestions for how to make these quantitative comparisons and suggest new molecular mechanisms that may allow non-equilibrium riboswitches to surpass the derived limit.
Topics: Aptamers, Nucleotide; Biosensing Techniques; Ligands; Nucleic Acid Conformation; RNA Folding; Riboswitch; Thermodynamics
PubMed: 31239056
DOI: 10.1016/bs.mie.2019.05.028 -
Cold Spring Harbor Perspectives in... Oct 2018The 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... (Review)
Review
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.
Topics: Animals; Humans; Models, Chemical; RNA; RNA Folding; Thermodynamics
PubMed: 30275276
DOI: 10.1101/cshperspect.a032433