RESUMEN
Guanidinium ion is a toxic cellular metabolite. The ykkC-III riboswitch, an mRNA stretch, regulates the gene expression by undergoing a conformational change in response to the binding of a free guanidinium ion and thereby plays a potentially important role in alleviating guanidinium toxicity in cells. An experimental crystal structure of the guanidinium-bound aptamer domain of the riboswitch from Thermobifida Fusca revealed the overall RNA architecture and mapped the specific noncovalent interactions that stabilize the ligand within the binding pocket aptamer. However, details of how the aptamer domain discriminates the cognate ligand from its closest structurally analogous physiological metabolites (arginine and urea), and how the binding of cognate ligand arrays information from the aptamer domain to the expression platform for regulating the gene expression, are not well understood. To fill this void, we perform a cumulative of 2 µs all-atom explicit-solvent molecular dynamics (MD) simulations on the full aptamer domain, augmented with quantum-chemical calculations on the ligand-binding pocket, to compare the structural and dynamical details of the guanidinium-bound state with the arginine or urea bound states, as well as the unbound (open) state. Analysis of the ligand-binding pocket reveals that due to unfavorable interactions with the binding-pocket residues, urea cannot bind the aptamer domain and thereby cannot alter the gene expression. Although interaction of the guanidyl moiety of arginine within the binding pocket is either comparable or stronger than the guanidinium ion, additional non-native hydrogen-bonding networks, as well as differences in the dynamical details of the arginine-bound state, explain why arginine cannot transmit the information from the aptamer domain to the expression platform. Based on our simulations, we propose a mechanism of how the aptamer domain communicates with the expression platform. Overall, our work provides interesting insights into the ligand recognition by a specific class of riboswitches and may hopefully inspire future studies to further understand the gene regulation by riboswitches.
Asunto(s)
Riboswitch , Guanidina , Ligandos , Simulación de Dinámica Molecular , Conformación de Ácido NucleicoRESUMEN
Transfer RNA (tRNA) is the most diversely modified RNA. Although the strictly conserved purine position 37 in the anticodon stem-loop undergoes modifications that are phylogenetically distributed, we do not yet fully understand the roles of these modifications. Therefore, molecular dynamics simulations are used to provide molecular-level details for how such modifications impact the structure and function of tRNA. A focus is placed on three hypermodified base families that include the parent i6A, t6A, and yW modifications, as well as derivatives. Our data reveal that the hypermodifications exhibit significant conformational flexibility in tRNA, which can be modulated by additional chemical functionalization. Although the overall structure of the tRNA anticodon stem remains intact regardless of the modification considered, the anticodon loop must rearrange to accommodate the bulky, dynamic hypermodifications, which includes changes in the nucleotide glycosidic and backbone conformations, and enhanced or completely new nucleobase-nucleobase interactions compared to unmodified tRNA or tRNA containing smaller (m1G) modifications at the 37th position. Importantly, the extent of the changes in the anticodon loop is influenced by the addition of small functional groups to parent modifications, implying each substituent can further fine-tune tRNA structure. Although the dominant conformation of the ASL is achieved in different ways for each modification, the molecular features of all modified tRNA drive the ASL domain to adopt the functional open-loop conformation. Importantly, the impact of the hypermodifications is preserved in different sequence contexts. These findings highlight the likely role of regulating mRNA structure and translation.
Asunto(s)
Adenosina/análogos & derivados , Anticodón/química , Escherichia coli/genética , Procesamiento Postranscripcional del ARN , ARN de Transferencia de Lisina/química , ARN de Transferencia de Fenilalanina/química , Adenosina/metabolismo , Anticodón/genética , Anticodón/metabolismo , Emparejamiento Base , Secuencia de Bases , Escherichia coli/metabolismo , Isopenteniladenosina/química , Isopenteniladenosina/metabolismo , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Nucleósidos/química , Nucleósidos/metabolismo , ARN de Transferencia de Lisina/genética , ARN de Transferencia de Lisina/metabolismo , ARN de Transferencia de Fenilalanina/genética , ARN de Transferencia de Fenilalanina/metabolismoRESUMEN
Aptamers are functional nucleic acids that bind to a range of targets (small molecules, proteins or cells) with a high affinity and specificity. Chemically-modified aptamers are of interest because the incorporation of novel nucleobase components can enhance aptamer binding to target proteins, while fluorescent base analogues permit the design of functional aptasensors that signal target binding. However, since optimally modified nucleoside designs have yet to be identified, information about how to fine tune aptamer stability and target binding affinity is required. The present work uses molecular dynamics (MD) simulations to investigate modifications to the prototypical thrombin-binding aptamer (TBA), which is a 15-mer DNA sequence that folds into a G-quadruplex structure connected by two TT loops and one TGT loop. Specifically, we modeled a previously synthesized thymine (T) analog, namely 5-furyl-2'-deoxyuridine (5FurU), into each of the six aptamer locations occupied by a thymine base in the TT or TGT loops of unbound and thrombin bound TBA. This modification and aptamer combination were chosen as a proof-of-principle because previous experimental studies have shown that TBA displays emissive sensitivity to target binding based on the local environment polarity at different 5FurU modification sites. Our simulations reveal that the chemically-modified base imparts noticeable structural changes to the aptamer without affecting the global conformation. Depending on the modification site, 5FurU performance is altered due to changes in the local environment, including the modification site structural dynamics, degree of solvent exposure, stacking with neighboring bases, and interactions with thrombin. Most importantly, these changes directly correlate with the experimentally-observed differences in the stability, binding affinity and emissive response of the modified aptamers. Therefore, the computational protocols implemented in the present work can be used in subsequent studies in a predictive way to aid the fine tuning of aptamer target recognition for use as biosensors (aptasensors) and/or therapeutics.