Shape Similarity Measures for the Design of Small RNA Switches

Abstract

Conformational switching in the secondary structure of RNAs has recently attracted considerable attention, fostered by the discovery of ‘riboswitches’ in living organisms. These are genetic control elements that were found in bacteria and offer a unique regulation mechanism based on switching between two highly stable states, separated by an energy barrier between them. In riboswitches, the energy barrier is crossed by direct metabolite binding, which facilitates regulation by allosteric means. However, other event triggers can cause switching to occur, such as single-point mutations and slight variations in temperature. Examples of switches with these event triggers have already been reported experimentally in the past. Here, the goal is to computationally design small RNA switches that rely on these triggers. Towards this end, our computer simulations utilize a variety of different similarity measures to assess the distances between an initial state and triggered states, based on the topology of the secondary structure itself. We describe these combined similarity measures that rely on both coarse-grained and fine-grained graph representations of the RNA secondary structure. As a result of our simulations, we provide some candidate sequences of approximately 30–50 nt, along with the exact triggers that drive the switching. The event triggers under consideration can be modelled by Zuker's mfold or the Vienna package. The proposed methodology that rely on shape measures can further be used to computationally generate more candidates by simulating various event triggers and calculating their effect on the shape.     

Folding path of P5abc RNA involves direct coupling of secondary and tertiary structures

Folding mechanisms in which secondary structures are stabilized through the formation of tertiary interactions are well documented in protein folding but challenge the folding hierarchy normally assumed for RNA. However, it is increasingly clear that RNA could fold by a similar mechanism. P5abc, a small independently folding tertiary domain of the Tetrahymena thermophila group I ribozyme, is known to fold by a secondary structure rearrangement involving helix P5c. However, the extent of this rearrangement and the precise stage of folding that triggers it are unknown. We use experiments and simulations to show that the P5c helix switches to the native secondary structure late in the folding pathway and is directly coupled to the formation of tertiary interactions in the A-rich bulge. P5c mutations show that the switch in P5c is not rate-determining and suggest that non-native interactions in P5c aid folding rather than impede it. Our study illustrates that despite significant differences in the building blocks of proteins and RNA, there may be common ways in which they self-assemble.     

Allosteric activation via kinetic control: potassium accelerates a conformational change in IMP dehydrogenase

Allosteric activators are generally believed to shift the equilibrium distribution of enzyme conformations to favor a catalytically productive structure; the kinetics of conformational exchange is seldom addressed. Several observations suggested that the usual allosteric mechanism might not apply to the activation of IMP dehydrogenase (IMPDH) by monovalent cations. Therefore, we investigated the mechanism of K(+) activation in IMPDH by delineating the kinetic mechanism in the absence of monovalent cations. Surprisingly, the K(+) dependence of k(cat) derives from the rate of flap closure, which increases by ≥65-fold in the presence of K(+). We performed both alchemical free energy simulations and potential of mean force calculations using the orthogonal space random walk strategy to computationally analyze how K(+) accelerates this conformational change. The simulations recapitulate the preference of IMPDH for K(+), validating the computational models. When K(+) is replaced with a dummy ion, the residues of the K(+) binding site relax into ordered secondary structure, creating a barrier to conformational exchange. K(+) mobilizes these residues by providing alternate interactions for the main chain carbonyls. Potential of mean force calculations indicate that K(+) changes the shape of the energy well, shrinking the reaction coordinate by shifting the closed conformation toward the open state. This work suggests that allosteric regulation can be under kinetic as well as thermodynamic control.     

An algorithm for searching RNA motifs in genomic sequences

RNA molecules, which are found in all living cells, fold into characteristic structures that account for their diverse functional activities. Many of these RNA structures consist of a collection of fundamental RNA motifs. The various combinations of RNA basic components form different RNA classes and define their unique structural and functional properties. The availability of many genome sequences makes it possible to search computationally for functional RNAs. Biological experiments indicate that functional RNAs have characteristic RNA structural motifs represented by specific combinations of base pairings and conserved nucleotides in the loop regions. The searching for those well-ordered RNA structures and their homologues in genomic sequences is very helpful for the understanding of RNA-based gene regulation. In this paper, we consider the following problem: given an RNA sequence with a known secondary structure, efficiently determine candidate segments in genomic sequences that can potentially form RNA secondary structures similar to the given RNA secondary structure. Our new bottom-up approach searches all potential stem-loops similar to ones of the given RNA secondary structure first, and then based on located stem-loops, detects potential homologous structural RNAs in genomic sequences.     

Design principles of protein switches

Protein switches perform essential roles in many biological processes and are exciting targets for de novo protein design, which aims to produce proteins of arbitrary shape and functionality. However, the biophysical requirements for switch function — multiple conformational states, fine-tuned energetics, and stimuli-responsiveness — pose a formidable challenge for design by computation (or intuition). A variety of methods have been developed toward tackling this challenge, usually taking inspiration from the wealth of sequence and structural information available for naturally occurring protein switches. More recently, modular switches have been designed computationally, and new methods have emerged for sampling unexplored structure space, providing promising new avenues toward the generation of purpose-built switches and de novo signaling systems for cellular engineering.     

Computational design and experimental validation of oligonucleotide-sensing allosteric ribozymes

Allosteric RNAs operate as molecular switches that alter folding and function in response to ligand binding. A common type of natural allosteric RNAs is the riboswitch; designer RNAs with similar properties can be created by RNA engineering. We describe a computational approach for designing allosteric ribozymes triggered by binding oligonucleotides. Four universal types of RNA switches possessing AND, OR, YES and NOT Boolean logic functions were created in modular form, which allows ligand specificity to be changed without altering the catalytic core of the ribozyme. All computationally designed allosteric ribozymes were synthesized and experimentally tested in vitro. Engineered ribozymes exhibit >1,000-fold activation, demonstrate precise ligand specificity and function in molecular circuits in which the self-cleavage product of one RNA triggers the action of a second. This engineering approach provides a rapid and inexpensive way to create allosteric RNAs for constructing complex molecular circuits, nucleic acid detection systems and gene control elements.     

The hydrodynamic shape, conformation, and molecular model of Escherichia coli ribosomal 5 S RNA.

The structure of ribosomal 5 S RNA has been examined using several physical biochemical techniques. Hydrodynamic measurements yield a s020,omega and [eta] of 5.5 x 10(-13) x and 6.9 ml/g, respectively. Other parameters calculated from these values indicate the shape of 5 S RNA is consistent with that of a prolate ellipsoid 160 A in length and 32 A wide. Sedimentation equilibrium results show that 5 S RNA exists as a monomer in the reconstitution buffer with an apparent molecular weight of 44,000. Ultraviolet absorption difference spectra show that approximately 75% of the bases in 5 S RNA are involved in base pairing, and of these base pairs 70% are G-C and 30% are A-U. These results on the overall shape and secondary structure of 5 S RNA have been incorporated with the results of other investigators as to the possible location of single-stranded and double-stranded helical regions, and a molecular model for 5 S RNA is proposed. The molecular model consists of three double helices in the shape of a prolate ellipsoid, with two of the double helical regions at one end of the molecule. The structure is consistent with the available data on the structure and function of 5 S RNA and bears similarity to the molecular model proposed by Osterberg et al. ((1976) Eur. J. Biochem. 68, 481-487) based on small angle x-ray scattering results and the secondary structure proposed by Madison ((1968) Annu. Rev. Biochem. 37, 131-148).     

Characterization of the unfolding pathway of the cell-cycle protein p13suc1 by molecular dynamics simulations: implications for domain swapping

BACKGROUND: The p13suc1 gene product is a member of the cks (cyclin-dependent protein kinase subunit) protein family and has been implicated in regulation of the cell cycle. Various crystal structures of suc1 are available, including a globular, monomeric form and a beta-strand exchanged dimer. It has been suggested that conversions between these forms, and perhaps others, may be important in the regulation of the cell cycle. RESULTS: We have undertaken molecular dynamics simulations of protein unfolding to investigate the conformational properties of suc1. Unfolding transition states were identified for each of four simulations. These states contain some native secondary structure, primarily helix alpha1 and the core of the beta sheet. The hydrophobic core is loosely packed. Further unfolding leads to an intermediate state that is slightly more expanded than the transition state, but with considerably fewer nonlocal, tertiary packing contacts and less secondary structure. The helices are fluctuating but partially formed in the denatured state and beta2 and beta4 remain associated. CONCLUSIONS: It appears that suc1 folds by a nucleation-condensation mechanism, similar to that observed for two-state folding proteins. However, suc1 forms an intermediate during unfolding and contains considerable residual structure in the denatured state. The stability of the beta2-beta4 residual structure is surprising, because beta4 is the strand involved in domain swapping. This stability suggests that the domain-swapping event, if physiologically relevant, may require the assistance of additional factors in vivo or occur early in the folding process.     

Function annotation for pseudoknot using structure similarity

Many raw biological sequence data have been generated by the human genome project and related efforts. The understanding of structural information encoded by biological sequences is important to acquire knowledge of their biochemical functions but remains a fundamental challenge. Recent interest in RNA regulation has resulted in a rapid growth of deposited RNA secondary structures in varied databases. However, a functional classification and characterization of the RNA structure have only been partially addressed. This article aims to introduce a novel interval-based distance metric for structure-based RNA function assignment. The characterization of RNA structures relies on distance vectors learned from a collection of predicted structures. The distance measure considers the intersected, disjoint, and inclusion between intervals. A set of RNA pseudoknotted structures with known function are applied and the function of the query structure is determined by measuring structure similarity. This not only offers sequence distance criteria to measure the similarity of secondary structures but also aids the functional classification of RNA structures with pesudoknots.     

Nodavirus RNA Recombination

The genomes of nodaviruses contain two positive-sense RNAs that encode the RNA polymerase and capsid proteins, respectively. In this system, recombination occurs when the polymerase switches templates during negative strand RNA synthesis, usually at a site where the nascent strand can form 4–5 bp with the acceptor template. Two other factors influence the choice of recombination site: (1) template secondary structure, which is predicted to hold the recombination sites in close proximity; and (2) similarity of the cross-over site to an origin of replication, which suggests that the polymerase interacts directly with the acceptor template.     

Second eigenvalue of the Laplacian matrix for predicting RNA conformational switch by mutation

MOTIVATION: Conformational switching in RNAs is thought to be of fundamental importance in several biological processes, including translational regulation, regulation of self-cleavage in viruses, protein biosynthesis and mRNA splicing. Current methods for detecting bi-stable RNAs that can lead to structural switching when triggered by an outside event rely on kinetics, energetics and properties of the combinatorial structure space of RNAs. Based on these properties, tools have been developed to predict whether a given sequence folds to a structure characterized by a bi-stable conformation, or to design multi-stable RNAs by an iterative algorithm. A useful addition is in developing a local procedure to prescribe, given an initial sequence, the least amount of mutations needed to drive the system into an optimal bi-stable conformation. RESULTS: We introduce a local procedure for predicting mutations, by generating and analyzing eigenvalue tables, that are capable of transforming the wild-type sequence into a bi-stable conformation. The method is independent of the folding algorithms but relies on their success. It can be used in conjunction with existing tools, as well as being incorporated into more general RNA prediction packages. We apply this procedure on three well-studied structures. First, the method is validated on the mutation leading to a conformational switch in the spliced leader RNA from Leptomonas collosoma, a mutation that has already been confirmed by an experiment. Second, the method is used to predict a mutation that can lead to a novel conformational switch in the P5abc subdomain of the group I intron ribozyme in Tetrahymena thermophila. Third, the method is applied on Hepatitis delta virus to predict mutations that transform the wild-type into a bi-stable conformation, a configuration assessed by calculating the free energies using folding prediction algorithms. The predictions in the final examples need to be verified experimentally, whereas the mutation predicted in the first example complies with the experiment. This supports the use of our proposed method on other known structures, as well as genetically engineered ones. AVAILABILITY: An eigenvalue application will be available in the near future attached to one of the existing tools.