Molecular dynamics simulation study of the binding of purine bases to the aptamer domain of the guanine sensing riboswitch

Riboswitches are a novel class of genetic control elements that function through the direct interaction of small metabolite molecules with structured RNA elements. The ligand is bound with high specificity and affinity to its RNA target and induces conformational changes of the RNA's secondary and tertiary structure upon binding. To elucidate the molecular basis of the remarkable ligand selectivity and affinity of one of these riboswitches, extensive all-atom molecular dynamics simulations in explicit solvent (≈1 μs total simulation length) of the aptamer domain of the guanine sensing riboswitch are performed. The conformational dynamics is studied when the system is bound to its cognate ligand guanine as well as bound to the non-cognate ligand adenine and in its free form. The simulations indicate that residue U51 in the aptamer domain functions as a general docking platform for purine bases, whereas the interactions between C74 and the ligand are crucial for ligand selectivity. These findings either suggest a two-step ligand recognition process, including a general purine binding step and a subsequent selection of the cognate ligand, or hint at different initial interactions of cognate and noncognate ligands with residues of the ligand binding pocket. To explore possible pathways of complex dissociation, various nonequilibrium simulations are performed which account for the first steps of ligand unbinding. The results delineate the minimal set of conformational changes needed for ligand release, suggest two possible pathways for the dissociation reaction, and underline the importance of long-range tertiary contacts for locking the ligand in the complex

Exploring RNA structure and dynamics through enhanced sampling simulations

RNA function is intimately related to its structural dynamics. Molecular dynamics simulations are useful for exploring biomolecular flexibility but are severely limited by the accessible timescale. Enhanced sampling methods allow this timescale to be effectively extended in order to probe biologically relevant conformational changes and chemical reactions. Here, we review the role of enhanced sampling techniques in the study of RNA systems. We discuss the challenges and promises associated with the application of these methods to force-field validation, exploration of conformational landscapes and ion/ligand-RNA interactions, as well as catalytic pathways. Important technical aspects of these methods, such as the choice of the biased collective variables and the analysis of multi-replica simulations, are examined in detail. Finally, a perspective on the role of these methods in the characterization of RNA dynamics is provided

Biomolecular simulations, from RNA to protein : thermodynamic and dynamic aspects

The process of transforming the information stored in the DNA of genes into functional RNA molecules and proteins via transcription and translation is the most fundamental process of all known life. Even though these processes involve large macromolecules and dynamics on long time scales they all ultimately rely on atomic level interactions between nucleic acids or amino acids. Only a few experimental techniques are available that can study the large systems involved in atomic detail. Computer simulations, modeling biological macromolecules, are therefore an important tool in investigating fundamental biological processes. In this thesis, Molecular Dynamics (MD) simulations have been used to study the translation of mRNA by tRNA and the function of the regulatory riboswitches. The thesis also covers the improvement of methodology by the development of a new representation of the important Mg2+ ions and an improvement of the understanding of the connection between MD and experimental NMR data. In Paper I, the effect of post transcriptional modifications of the tRNA anti codon on the decoding of mRNA in the ribosome is studied. All atom MD simulations have been performed of the ribosomal A site with and without modifications present, including extensive free energy calculations. The results show two mechanism by which the decoding is affected: The further reach provided by the modifications allows an alternative outer conformation to be formed for the non cognate base pairs, and the modifications results in increased “catalytic” contacts between tRNA, mRNA and the ribosome. In Paper II, the folding mechanism of the add A riboswitch is studied under different ionic conditions and with and without the ligand bound. In addition to standard simulations, we simulated the unfolding by umbrella sampling of distance between the L2 and L3 loops. In the results, no significant effect of Mg2+ or Na+ ion environments or ligand presence can be seen. But a consistent mechanism with the P3 stem being more flexible than P2 is observed. More data might however be needed to draw general conclusions. In Paper III, the parameters describe Mg2+ ions in MD simulations are improved by optimizing to kinetic data of the H2O exchange. Data from NMR relaxation experiments was used as optimization goal. The newly developed parameters do not only display better kinetic properties, but also better agreement with experimental structural data. In Paper IV, the dynamical data, obtained from NMR relaxation experiment of a protein is related to dynamics seen in an MD simulation. The analysis provides important information for the interpretation of experimental data and the development of simulation methods. The results show, among other things, that significant parts of the entropy are not seen by NMR due to a limited time window and inability to account for correlation of motions

Study of complex RNA function modulated by small molecules: the development of RNA directed small molecule library and probing the S-adenosyl methionine discrimination between on and off conformational states of the SAM-I riboswitch

RNA recently remained unexploited and is now drawing interest as a potential drug target. The methodology and available drug libraries for RNA targeting/screening are in rudimentary stages. The interactions made by ligands with RNA can be explored for RNA based drug development. The dissertation is composed of 4 chapters. The first chapter focuses on the structural features of RNA and the attempts made to target RNA previously. The second chapter focuses on the development of a small molecule library enriched with substructures derived from RNA binding ligands. For this study a fragment-based approach (fragment based approach is detailed in chapter 2) is used in order to accommodate the conformational flexibility of RNA. The library molecules are used for screening against suitable RNA targets using NMR. We identified at least 5 ligands out of which 2 are novel ligands binding to the ribosomal 16s rRNA. The third chapter is focused on the role of small molecules in inducing conformational changes in an RNA genetic regulatory element called the S-Adenosyl methionine (SAM) SAM-I riboswitch. The mechanistic features of the SAM-I riboswitch to understand the basis for specificity and discrimination and its gene regulation mechanism are reported. To address the conformational dynamics Bacillus subtilis and Thermoanearobacter tencongenesis SAM-I riboswitches in response to SAM binding several conformer mimics are designed, synthesized and characterized using NMR, equilibrium dialysis, and inline probing. The study shows that apart from the conserved residues of the binding pocket, residues downstream of the binding pocket are involved in detecting SAM and assist the binding of SAM to the riboswitch with weak affinity. Our data highlights the capacity of a so-called antiterminator helix from the expression platform to assist the formation of a partial P1 helix of the aptamer domain. A stable P1 is involved in recognition and tight binding of SAM. Our in vitro experiments suggest that the riboswitch could switch from an unbound conformation to tightly SAM bound structure through weakly binding intermediate structures in the presence of the small molecule SAM. The future directions are included in the fourth chapter along with the conclusions

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

Deciphering the Details of RNA Aminoglycoside Interactions: From Atomistic Models to Biotechnological Applications

Aminoglycosides are a class of antibiotics functioning through binding to 16S rRNA A-site and inhibiting the bacterial translation. However, the continuous emergence of drug-resistant strains makes the development of new and more potent antibiotics necessary. Aminoglycosides are also known to interact with various biologically crucial RNA molecules other than 16S rRNA A-site and inhibit their functions. As a result, they are considered as the single most important model to understand the principles of RNA small molecule recognition. The detailed understanding of these interactions is necessary for the development of novel antibacterial, antiviral or even anti-oncogenic agents. In our studies, we have studied both the natural aminoglycoside targets like Rev responsive element (RRE), trans-activating region (TAR) of HIV-1 and thymidylate synthase mRNA 5\u27 untranslated (UTR) region as well as the in vitro selected neomycin, tobramycin and kanamycin RNA aptamers. By this way, we think we have covered a variety of binding pockets to figure out the critical nucleic acid residues playing essential role in aminoglycoside recognition. Along with all these RNAs, we studied more than 10 aminoglycoside ligands to pinpoint the chemical groups in close contact with RNAs. To determine thermodynamic parameters for these interactions, we utilized isothermal titration calorimetry (ITC) assay by which we found that the majority of these interactions are enthalpy driven. More specifically, RNA aminoglycoside interactions are mainly derived by electrostatic and hydrogen binding interactions. Our studies indicated that the amino groups on the first ring of the aminoglycosides are essential for high affinity binding whereas having bulky groups on ring II sterically eliminate their interactions with RNAs. RNA binding trend of aminoglycosides are as follows: neomycin-B \u3e ribostamycin \u3e kanamycin-B \u3e tobramycin \u3e paromomycin \u3e sisomicin \u3e gentamicin \u3e kanamycin-A \u3e geneticin \u3e amikacin \u3e netilmicin. Aminoglycoside binding to the aptamer was shown highly buffer dependent. This phenomenon was analyzed in five different buffers and found that cacodylate-based buffer changes the specificity of the aptamer. In addition to ITC, we have used molecular docking to specifically find out the chemical groups in these interactions. We have specified the nucleic acid residues interacting with aminoglycosides. In parallel, molecular dynamics (MD) simulations of neomycin RNA aptamer with neomycin-B in an all-atom platform in GROMACS were carried out. The results showed a mobile structure consistent with the ability of this aptamer to interact with a wide range of ligands. From molecular docking and MD simulations, we identified the neomycin-B aptamer residues that might contribute to its ligand selectivity and designed a series of new aptamers accordingly. Also, A16 was found to be flexible, which was confirmed by 2AP fluorescence studies. In this analysis, the buffer dependence was also confirmed against neomycin-B, ribostamycin and paromomycin. One of the challenges in therapeutics is the emergence of resistant cells. They become reistant to the drugs via changing the target site, or enzymatically modifying the drug, or producing drug pumps to export the drugs. To overcome the very last challenge, we are utilizing RNA-aminoglycoside partners to keep high intracellular drug concentration and increase the efficacy of aminoglycosides against bacteria. We called the system as DRAGINs (Drug binding aptamers for growing intracellular numbers). We express these RNAs in bacteria and detect their growth rate in order to evaluate their response to different concentration of aminoglycosides. In this study, we found that we could successfully decrease the IC50 values by 2 to 5 fold with the help of aminoglycoside-binding RNA aptamers. Finally, we are mathematically modeling the effect of aptamers on IC50 values of drugs with the use of four-compartment model. In our research group, we are utilizing these RNA-aminoglycoside partners to develop tags for detecting RNA in vivo and in real time. We called this system as intracellular multiaptamer genetic tags (IMAGEtags)

NMR-Untersuchungen zu dynamischen Umfaltungsprozessen in RNA-Molekülen

The following thesis is concerned with the elucidation of structural changes of RNA molecules during the time course of dynamic processes that are commonly denoted as folding reactions. In contrast to the field of protein folding, the concept of RNA folding comprises not only folding reactions itself but also refolding- or conformational switching- and assembly processes (see chapter III). The method in this thesis to monitor these diverse processes is high resolution liquid-state NMR spectroscopy. To understand the reactions is of considerable interest, because most biological active RNA molecules function by changing their conformation. This can be either an intrinsic property of their respective sequence or may happen in response to a cellular signal such as small molecular ligand binding (like in the aptamer and riboswitch case), protein or metal binding. The first part of the thesis (chapters II & III) provides a general overview over the field of RNA structure and RNA folding. The two chapters aim at introducing the reader into the current status of research in the field. Chapters II is structured such that primary structure is first described then secondary and tertiary structure elements of RNA structure. A special emphasis is given to bistable RNA systems that are functionally important and represent models to understand fundamental questions of RNA conformational switching. RNA folding in vitro as well as in vivo situations is discussed in Chapter III. The following chapters IV and V also belong to the introduction part and review critically the NMR methods that were used to understand the nature and the dynamics of the conformational/structural transitions in RNA. A general overview of NMR methods quantifying dynamics of biomolecules is provided in chapter IV. A detailed discussion of solvent exchange rates and time-resolved NMR, as the two major techniques used, follows. In the final chapter V of the first part the NMR parameters used in structure calculation and structure calculation itself are conferred. The second part of the thesis, which is the cumulative part, encompasses the conducted original work. Chapter VI reviews the general NMR techniques applied and explains their applicability in the field of RNA structural and biochemical studies in several model cases. Chapter VII describes the achievement of a complete resonance assignment of an RNA model molecule (14mer cUUCGg tetral-loop RNA) and introduces a new technique to assign quaternary carbon resonances of the nucleobases. Furthermore, it reports on a conformational analysis of the sugar backbone in this RNA hairpin molecule in conjunction with a parameterization of 1J scalar couplings. Achievements: • Establishment of two new NMR pulse-sequences facilitating the assignment of quaternary carbons in RNA nucleobases • First complete (99.5%) NMR resonance assignment of an RNA molecule (14mer) including 1H, 13C, 15N, 31P resonances • Description of RNA backbone conformation by a complete set of NMR parameters • Description of the backbone conformational dependence in RNA of new NMR parameters (1J scalar couplings) Chapters VII & VIII summarize the real-NMR studies that were conducted to elucidate the conformational switching events of several RNA systems. Chapter VIII gives an overview on the experiments that were accomplished on three different bistable RNAs. These molecules where chosen to be good model systems for RNA refolding reactions and so consequently served as reporters of conformational switching events of RNA secondary structure elements. Achievements: • First kinetic studies of RNA refolding reactions with atomic resolution by NMR • Application of [new] RT-NMR techniques either regarding the photolytic initiation of the reaction or regarding the readout of the reaction • Discovery of different RNA refolding mechanisms for different RNA molecules Deciphering of a general rule for RNA refolding methodology to conformational switching processes of RNA tertiary structure elements. The models for these processes were a) the guanine-dependent riboswitch RNA and b) the minimal hammerhead ribozyme. Achievements: • NMR spectroscopic assignment of imino-resonances of the hypoxanthine bound guanine-dependent riboswitch RNA • Application of RT-NMR techniques to monitor the ligand induced conformational switch of the aptamer domain of the guanine-dependent riboswitch RNA at atomic resolution • Translation of kinetic information into structural information • Deciphering a folding mechanism for the guanine riboswitch aptamer domain • Application of RT-NMR techniques to monitor the reaction of the catalytically active mHHR RNA at atomic resolution In the appendices the new NMR pulse-sequences and the experimental parameters are described, which are not explicitly treated in the respective manuscripts.Die vorliegende Doktorarbeit beschäftigt sich mit den strukturellen Änderungen in RNA Molekülen während dynamischer konformationeller Änderungen, die gemeinhin als RNA-Faltung bezeichnet werden. Im Gegensatz zur Proteinfaltung sind RNA-Faltungsprozesse nicht exklusiv als die Faltung einer definierten Konformation aus einem Ensemble an ungefalteten, d.h. ausgehend von unstrukturierten Molekülen, zu verstehen. RNA-Faltung beinhaltet vielmehr die strukturelle Umwandlung verschiedener stabiler Konformationen (die als RNA-Umfaltung benannt wird) und den Aufbau von molekularen Komplexen aus mehreren Molekülen (siehe Kapitel III). Die experimentelle Technik, die hier zur Untersuchung dieser Prozesse genutzt wurde, ist die hochauflösende Flüssig-NMR-Spektroskopie. Das Verständnis der strukturellen und biophysikalischen Grundlagen solcher Umfaltungsreaktionen von RNA ist essentiell, da solche konformationellen Änderungen die biologische Funktion der Moleküle modulieren. Dabei ist zu bemerken, dass eine Umfaltungsreaktion eine intrinsische Eigenschaft einer gegebenen RNA-Sequenz sein kann oder die Antwort auf ein externes zelluläres Signal, wie die Bindung eines niedermolekularen Liganden (z.B. in Aptameren und in Riboswitch RNAs), eines Proteins oder eines Metall-Ions. Der erste Teil dieser Doktorarbeit (Kapitel I & II) hält einen Überblick über die Themengebiete RNA-Struktur und RNA-Faltung bereit. Beide Kapitel führen in den derzeitigen Stand der Forschung ein. Kapitel II führt dabei entlang der hierarchischen Ordnung von RNA Molekülen und diskutiert die Eigenschaften von Primär-, Sekundär- und Tertiär-Strukturelementen. Ein besonderes Augenmerk wird dabei auf bistabile RNA Systeme gelegt; ihre wichtige biologische Funktionalität wird dargestellt, ebenso wird das Potential ausgeleuchtet, diese funktionale Klasse von RNA Molekülen als Modellsysteme zu nutzen, um fundamentale Fragen zu konformationellen Übergängen in RNA zu beantworten. In Kapitel III folgt sodann die Diskussion über RNA-Faltung in in vitro Experimenten als auch im zellulären Kontext (in vivo). Die Kapitel IV und V besprechen die NMR-spektroskopischen Techniken, die genutzt werden, um die Art und die dynamischen Eigenschaften von konformationellen/strukturellen Umwandlungen in RNA zu untersuchen. Hierbei wird der Schwerpunkt auf die verwendeten Techniken des Wasseraustauschs an labilen Protonen und der zeitaufgelösten NMR-Spektroskopie gelegt. Der zweite Teil der Doktorarbeit fasst kumulativ die durchgeführten Studien zusammen. Kapitel VI bespricht hierbei die grundlegenden NMR Techniken, die zur Strukturaufklärung von RNA Molekülen angewendet werden und zeigt deren Anwendungsmöglichkeiten an unterschiedlichen Beispielen von strukturellen und biochemischen Studien. Das folgende Kapitel VII beschreibt die komplette Resonanzzuordnung eines RNA Modell-Moleküls (14mer cUUCGg tetra-loop RNA) und stellt eine neue Pulstechnik vor, die zur Zuordnung der Resonanzen von quatären Kohlenstoffen in Purinbasen benützt werden kann. Weiterhin schließt sich ein Report an, wie die Konformation des Zuckerrückgrates in RNA-Molekülen bestimmt wird und schlägt mittels einer an oben genanntem Modellsystem durchgeführte Parametrisierung 1J skalare Kopplungen als neue Strukturparameter vor. Kapitel VII & VIII fassen die hierzu durchgeführten RT-NMR Studien zusammen. Kapitel VIII gibt hierbei einen Überblick über die Untersuchungen an drei bistabilen RNA-Systemen. Diese Moleküle wurden ausgewählt, da sie als Modelle für RNA-Umfaltungsreakionen dienen. Das finale Kapitel IX behandelt die Anwendung der oben ausgeführten neuen Methodologie auf konformationelle Umwandlungen von RNA Tertiär-Strukturelementen: a) Guanin-abhängige Riboswitch RNA (GSW) und b) Minimales "hammerhead" Ribozym (mHHR)

An excited state underlies gene regulation of a transcriptional riboswitch

Riboswitches control gene expression through ligand-dependent structural rearrangements of the sensing aptamer domain. However, we found that the Bacillus cereus fluoride riboswitch aptamer adopts identical tertiary structures in solution with and without ligand. Using chemical exchange saturation transfer (CEST) NMR spectroscopy, we revealed that the structured ligand-free aptamer transiently accesses a low-populated (~1%) and short-lived (~3 ms) excited conformational state that unravels a conserved ‘linchpin’ base pair to signal transcription termination. Upon fluoride binding, this highly localized fleeting process is allosterically suppressed to activate transcription. We demonstrated that this mechanism confers effective fluoride-dependent gene activation over a wide range of transcription rates, which is essential for robust toxicity response across diverse cellular conditions. These results unveil a novel switching mechanism that employs ligand-dependent suppression of an aptamer excited state to coordinate regulatory conformational transitions rather than adopting distinct aptamer ground-state tertiary architectures, exemplifying a new mode of ligand-dependent RNA regulation

Theoretical and computational modeling of rna-ligand interactions

Ribonucleic acid (RNA) is a polymeric nucleic acid that plays a variety of critical roles in gene expression and regulation at the level of transcription and translation. Recently, there has been an enormous interest in the development of therapeutic strategies that target RNA molecules. Instead of modifying the product of gene expression, i.e., proteins, RNAtargeted therapeutics aims to modulate the relevant key RNA elements in the disease-related cellular pathways. Such approaches have two significant advantages. First, diseases with related proteins that are difficult or unable to be drugged become druggable by targeting the corresponding messenger RNAs (mRNAs) that encode the amino acid sequences. Second, besides coding mRNAs, the vast majority of the human genome sequences are transcribed to noncoding RNAs (ncRNAs), which serve as enzymatic, structural, and regulatory elements in cellular pathways of most human diseases. Targeting noncoding RNAs would open up remarkable new opportunities for disease treatment. The first step in modeling the RNA-drug interaction is to understand the 3D structure of the given RNA target. With current theoretical models, accurate prediction of 3D structures for large RNAs from sequence remains computationally infeasible. One of the major challenges comes from the flexibility in the RNA molecule, especially in loop/junction regions, and the resulting rugged energy landscape. However, structure probing techniques, such as the “selective 20-hydroxyl acylation analyzed by primer extension” (SHAPE) experiment, enable the quantitative detection of the relative flexibility and hence structure information of RNA structural elements. Therefore, one may incorporate the SHAPE data into RNA 3D structure prediction. In the first project, we investigate the feasibility of using a machine-learning-based approach to predict the SHAPE reactivity from the 3D RNA structure and compare the machine-learning result to that of a physics-based model. In the second project, in order to provide a user-friendly tool for RNA biologists, we developed a fully automated web interface, “SHAPE predictoR” (SHAPER) for predicting SHAPE profile from any given 3D RNA structure. In a cellular environment, various factors, such as metal ions and small molecules, interact with an RNA molecule to modulate RNA cellular activity. RNA is a highly charged polymer with each backbone phosphate group carrying one unit of negative (electronic) charge. In order to fold into a compact functional tertiary structure, it requires metal ions to reduce Coulombic repulsive electrostatic forces by neutralizing the backbone charges. In particular, Mg2+ ion is essential for the folding and stability of RNA tertiary structures. In the third project, we introduce a machine-learning-based model, the “Magnesium convolutional neural network” (MgNet) model, to predict Mg2+ binding site for a given 3D RNA structure, and show the use of the model in investigating the important coordinating RNA atoms and identifying novel Mg2+ binding motifs. Besides Mg2+ ions, small molecules, such as drug molecules, can also bind to an RNA to modulate its activities. Motivated by the tremendous potential of RNA-targeted drug discovery, in the fourth project, we develop a novel approach to predicting RNA-small molecule binding. Specifically, we develop a statistical potential-based scoring/ranking method (SPRank) to identify the native binding mode of the small molecule from a pool of decoys and estimate the binding affinity for the given RNA-small molecule complex. The results tested on a widely used data set suggest that SPRank can achieve (moderately) better performance than the current state-of-art models

A Tale of Two RNAs: Single Molecule Investigation of the Conformation, Dynamics and Ligand Binding to the PreQ1 and T-box Riboswitches.

Riboswitches are structured mRNA domains that can bind cellular metabolites and control gene expression of downstream genes mainly via transcription attenuation or inhibition of translation initiation. Although structures of many ligand-bound riboswitches are available, knowledge on their ligand-free conformations is scarce. Subsequently, the ligand-mediated folding process of riboswitches is poorly understood. In this dissertation, we used single molecule FRET to investigate the conformation and ligand binding properties of two very distinct riboswitches. We showed that, contrary to previous studies, the structurally similar but functionally different preQ1 riboswitches from B. subtilis (Bsu) and T. tencongensis (Tte) have similar conformational ensemble in their ligand-free state with only subtle differences in their dynamics. Our smFRET data in combination with computational simulations suggested that both the riboswitches adopt ligand-free ‘pre-folded’ conformations and fold through distinct pathways that are similar to the conformational selection and induced fit mechanisms, respectively. We also demonstrated how remote mutations can affect the ligand binding affinities of riboswitches. Later, using smFRET, we probe the effect of various ligands on the kinetics of the Bsu riboswitch conformational dynamics with an aim to dissect its ligand binding mechanism. Our data suggest that the Bsu riboswitch can fold through both induced fit or conformational selection pathways, the relative extent of which is dependent on the presence of Mg2+. The T-box riboswitch is one of the complex riboswitches that binds tRNA and controls gene expression by sensing the relative levels of charged and uncharged tRNA. The structure of a T-box riboswitch stem-I:tRNA complex was recently solved, but it lacks the important genetic regulatory domain. By using various designs of the glyQS T-box riboswitch, we have studied the global conformation of the full T-box riboswitch and estimated distances between different regions. We measured tRNA binding kinetics to different T-box variants and showed that the double T-loop motif only contributes modestly to decrease the tRNA dissociation rate. Further, we directly demonstrated that the presence of glycine increases the tRNA dissociation rate ~6-fold that forms the basis of T-box riboswitch mechanism. Based on our kinetic data, we propose an improved kinetic model of the T-box riboswitch function.PHDBiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/108919/1/krishnac_1.pd