From RNA folding to inverse folding: a computational study: Folding and design of RNA molecules

Since the discovery of the structure of DNA in the early 1953s and its double-chained complement of information hinting at its means of replication, biologists have recognized the strong connection between molecular structure and function. In the past two decades, there has been a surge of research on an ever-growing class of RNA molecules that are non-coding but whose various folded structures allow a diverse array of vital functions. From the well-known splicing and modification of ribosomal RNA, non-coding RNAs (ncRNAs) are now known to be intimately involved in possibly every stage of DNA translation and protein transcription, as well as RNA signalling and gene regulation processes. Despite the rapid development and declining cost of modern molecular methods, they typically can only describe ncRNA's structural conformations in vitro, which differ from their in vivo counterparts. Moreover, it is estimated that only a tiny fraction of known ncRNAs has been documented experimentally, often at a high cost. There is thus a growing realization that computational methods must play a central role in the analysis of ncRNAs. Not only do computational approaches hold the promise of rapidly characterizing many ncRNAs yet to be described, but there is also the hope that by understanding the rules that determine their structure, we will gain better insight into their function and design. Many studies revealed that the ncRNA functions are performed by high-level structures that often depend on their low-level structures, such as the secondary structure. This thesis studies the computational folding mechanism and inverse folding of ncRNAs at the secondary level. In this thesis, we describe the development of two bioinformatic tools that have the potential to improve our understanding of RNA secondary structure. These tools are as follows: (1) RAFFT for efficient prediction of pseudoknot-free RNA folding pathways using the fast Fourier transform (FFT)}; (2) aRNAque, an evolutionary algorithm inspired by Lévy flights for RNA inverse folding with or without pseudoknot (A secondary structure that often poses difficulties for bio-computational detection). The first tool, RAFFT, implements a novel heuristic to predict RNA secondary structure formation pathways that has two components: (i) a folding algorithm and (ii) a kinetic ansatz. When considering the best prediction in the ensemble of 50 secondary structures predicted by RAFFT, its performance matches the recent deep-learning-based structure prediction methods. RAFFT also acts as a folding kinetic ansatz, which we tested on two RNAs: the CFSE and a classic bi-stable sequence. In both test cases, fewer structures were required to reproduce the full kinetics, whereas known methods (such as Treekin) required a sample of 20,000 structures and more. The second tool, aRNAque, implements an evolutionary algorithm (EA) inspired by the Lévy flight, allowing both local global search and which supports pseudoknotted target structures. The number of point mutations at every step of aRNAque's EA is drawn from a Zipf distribution. Therefore, our proposed method increases the diversity of designed RNA sequences and reduces the average number of evaluations of the evolutionary algorithm. The overall performance showed improved empirical results compared to existing tools through intensive benchmarks on both pseudoknotted and pseudoknot-free datasets. In conclusion, we highlight some promising extensions of the versatile RAFFT method to RNA-RNA interaction studies. We also provide an outlook on both tools' implications in studying evolutionary dynamics

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA-ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field

Mechanistic Insights into Molecular Recognition and the Regulatory Landscape of the lysine Riboswitch

RNA plays a central role in gene regulation and information processing in all kingdoms of life. Found in 5\u27 leader sequence of many bacterial mRNAs, riboswitches serve as an exemplar of this duality as they control expression of their own transcript by directly binding small molecule metabolites in the cell. These RNAs adopt tertiary structures to scaffold highly specific ligand binding sites, reminiscent of their protein counterparts. Ligand binding is then coupled to conformational changes in the RNA that influence expression of the downstream message by altering the transcription or translation of the message. To investigate the structural basis by which bacterial mRNAs specifically recognize lysine the aptamer domain from the leader sequence of the asd gene in Thermatoga maritima was solved by X-ray crystallography in both the free and bound conformations. These structures were complemented with solution based approaches to demonstrate that the tertiary architecture of the lysine aptamer domain is largely preorganized at 5 mM Mg2+ in the absence of ligand. Ligand binding was found to induce limited conformational changes within the five-way junction of the RNA. Based on these collective observations a site-specifically labeled RNA construct was designed to enable further thermodynamic and kinetic analysis of ligand binding to the aptamer. Using a series of lysine analogs that challenge key aspects of the structural model, we obtained a detailed understanding of the energetics of ligand recognition and demonstrate the importance of solvent and ion-mediated contacts in achieving a high affinity interaction. The binding kinetics of these analogs were also used to develop a simple mathematical framework for predicting the regulatory behavior of the RNA during transcription. Kinetic predictions were tested using a minimally reconstituted in vitro transcription assay to gain further empirical insight into the regulatory functions of the B. subtilis lysine riboswitch and correlate the biological function with studies of the isolated aptamer. The regulatory response of lysine was found to agree well with a simple two state mechanism of ligand binding for lysine at a variety of NTP concentrations. The five fold variation in T50 observed for lysine along with changes in the observed termination efficiency also suggest that this RNA by indirect means integrates a more global picture of metabolism into its regulatory response. Kinetic predictions were also predictive for the regulatory response of many of the alternative ligands at low NTP concentrations, but were found to be less accurate in predicting responses at elevated NTP concentrations, suggesting that the simple model may neglect certain features of the transcription process. The in vitro transcription assays were also employed to study the mechanism by which ligand binding is coupled to the secondary structural switch in the expression platform. A systematic survey of mutations to the P1 helix demonstrated that this element serves as the primary module for interdomain communication in the natural riboswitches, an insight that facilitated approaches to rationally design and optimize chimeric riboswitches. These studies have collectively shown that the regulatory switch is self contained in the expression platform and can be reprogrammed to be responsive to a large number of alternative ligands through a simple mix and match approach

Studying the dynamical properties of small RNA molecules with computational techniques

The role of ribonucleic acid (RNA) in molecular biology is shifting from a mere messenger between DNA (deoxyribonucleic acid) and proteins to an important player in many cellular activities. The central role of RNA molecules calls for a precise characterization of their structural and dynamical properties. Nowadays, experiments can be efficiently complemented by computational approaches. This thesis deals with the study of the dynamical properties of small RNA molecules, exploiting various computational techniques. Specifically we investigate two different complementary methods, elastic network models (ENMs) and Markov state models (MSMs). ENMs are valuable and efficient tools for characterizing the collective internal dynamics of biomolecules. We evaluate their performance by comparing their predictions with the results of atomistic molecular dynamics (MD) simulations and selective 2\u2019-hydroxyl analyzed by primer extension (SHAPE) experiments. We identify the optimal parameters that should be adopted when putting into use such models. MSMs are tools that allow to probe long-term molecular kinetics based on short-time MD simulations. We make use of MSMs and MD simulations to measure the kinetics and the timescale of the stacking-unstacking motion for a collection of short RNA oligonucleotides, comparing the results with previously published relaxation experiments. We then move to the study of the process of the fraying of the terminal base pair in a helix, characterizing the different involved pathways and the sequence dependence of the process timescale

Interactions in the cpSRP Dependent Targeting of Light Harvesting Chlorophyll Binding Protein to the Thylakoid Membrane

Targeting of proteins is a critical component of cellular function. A universally conserved targeting system of the cytosol utilizes a signal recognition particle (SRP) to target many proteins contranslationally to the endoplasmic reticulum in eukaryotes or the inner membrane in prokaryotes. A homologous SRP system exists in the chloroplast that delivers light harvesting chlorophyll binding proteins (LHCP) to they thylakoid membrane. The chloroplast SRP (cpSRP) is a heterodimer composed of a novel 43 kDa subunit and a 54 kDa subunit homologous to a component of the SRP system, SRP54. Many details regarding the interactions between the proteins of the cpSRP system have been determined. However, the three-dimensional arrangement of the cpSRP43 and cpSRP54 domains as well as their influence on one another has not been determined. The results of this study demonstrate both cpSRP43 and cpSRP54 are characterized by a significant amount of structural flexibility. Specifically, the domains of cpSRP43 and cpSRP54 are flexibly linked allowing for rapid conformational sampling of the domains. This flexibility allows cpSRP43 to sense the presence of cpSRP54 and subsequently alter its affinity for LHCP. Conversely, cpSRP54 domain flexibility allows it to scan cpSRP43 for the third transmembrane segment of LHCP in a manner surprisingly similar to SRP54 scanning for signal sequences at the ribosome. Together, the results of this structural investigation of the free and bound proteins has lead to the speculation of a cpSRP-LHCP transit complex structure capable of rationalizing the steps leading to the integration of LHCP