RESUMO
Riboswitches regulate downstream gene expression by binding cellular metabolites. Regulation of translation initiation by riboswitches is posited to occur by metabolite-mediated sequestration of the Shine-Dalgarno sequence (SDS), causing bypass by the ribosome. Recently, we solved a co-crystal structure of a prequeuosine1-sensing riboswitch from Carnobacterium antarcticum that binds two metabolites in a single pocket. The structure revealed that the second nucleotide within the gene-regulatory SDS, G34, engages in a crystal contact, obscuring the molecular basis of gene regulation. Here, we report a co-crystal structure wherein C10 pairs with G34. However, molecular dynamics simulations reveal quick dissolution of the pair, which fails to reform. Functional and chemical probing assays inside live bacterial cells corroborate the dispensability of the C10-G34 pair in gene regulation, leading to the hypothesis that the compact pseudoknot fold is sufficient for translation attenuation. Remarkably, the C. antarcticum aptamer retained significant gene-regulatory activity when uncoupled from the SDS using unstructured spacers up to 10 nucleotides away from the riboswitch-akin to steric-blocking employed by sRNAs. Accordingly, our work reveals that the RNA fold regulates translation without SDS sequestration, expanding known riboswitch-mediated gene-regulatory mechanisms. The results infer that riboswitches exist wherein the SDS is not embedded inside a stable fold.
Assuntos
Biossíntese de Proteínas , Riboswitch , Sítios de Ligação , Regulação da Expressão Gênica , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Riboswitches are small noncoding RNAs found primarily in the 5' leader regions of bacterial messenger RNAs where they regulate expression of downstream genes in response to binding one or more cellular metabolites. Such noncoding RNAs are often regulated at the translation level, which is thought to be mediated by the accessibility of the Shine-Dalgarno sequence (SDS) ribosome-binding site. Three classes (I-III) of prequeuosine1 (preQ1)-sensing riboswitches are known that control translation. Class I is divided into three subtypes (types I-III) that have diverse mechanisms of sensing preQ1, which is involved in queuosine biosynthesis. To provide insight into translation control, we determined a 2.30 Å-resolution cocrystal structure of a class I type III preQ1-sensing riboswitch identified in Escherichia coli (Eco) by bioinformatic searches. The Eco riboswitch structure differs from previous preQ1 riboswitch structures because it has the smallest naturally occurring aptamer and the SDS directly contacts the preQ1 metabolite. We validated structural observations using surface plasmon resonance and in vivo gene-expression assays, which showed strong switching in live E. coli. Our results demonstrate that the Eco riboswitch is relatively sensitive to mutations that disrupt noncanonical interactions that form the pseudoknot. In contrast to type II preQ1 riboswitches, a kinetic analysis showed that the type III Eco riboswitch strongly prefers preQ1 over the chemically similar metabolic precursor preQ0. Our results reveal the importance of noncanonical interactions in riboswitch-driven gene regulation and the versatility of the class I preQ1 riboswitch pseudoknot as a metabolite-sensing platform that supports SDS sequestration.
Assuntos
Riboswitch , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Pirimidinonas/química , RNA Bacteriano/genética , Conformação de Ácido Nucleico , LigantesRESUMO
The first RNA category of the Critical Assessment of Techniques for Structure Prediction competition was only made possible because of the scientists who provided experimental structures to challenge the predictors. In this article, these scientists offer a unique and valuable analysis of both the successes and areas for improvement in the predicted models. All 10 RNA-only targets yielded predictions topologically similar to experimentally determined structures. For one target, experimentalists were able to phase their x-ray diffraction data by molecular replacement, showing a potential application of structure predictions for RNA structural biologists. Recommended areas for improvement include: enhancing the accuracy in local interaction predictions and increased consideration of the experimental conditions such as multimerization, structure determination method, and time along folding pathways. The prediction of RNA-protein complexes remains the most significant challenge. Finally, given the intrinsic flexibility of many RNAs, we propose the consideration of ensemble models.
Assuntos
Biologia Computacional , Proteínas , Conformação Proteica , Proteínas/química , Modelos Moleculares , Biologia Computacional/métodos , Difração de Raios XRESUMO
Riboswitches are structured RNA motifs that recognize metabolites to alter the conformations of downstream sequences, leading to gene regulation. To investigate this molecular framework, we determined crystal structures of a preQ1-I riboswitch in effector-free and bound states at 2.00 Å and 2.65 Å-resolution. Both pseudoknots exhibited the elusive L2 loop, which displayed distinct conformations. Conversely, the Shine-Dalgarno sequence (SDS) in the S2 helix of each structure remained unbroken. The expectation that the effector-free state should expose the SDS prompted us to conduct solution experiments to delineate environmental changes to specific nucleobases in response to preQ1. We then used nudged elastic band computational methods to derive conformational-change pathways linking the crystallographically-determined effector-free and bound-state structures. Pathways featured: (i) unstacking and unpairing of L2 and S2 nucleobases without preQ1-exposing the SDS for translation and (ii) stacking and pairing L2 and S2 nucleobases with preQ1-sequestering the SDS. Our results reveal how preQ1 binding reorganizes L2 into a nucleobase-stacking spine that sequesters the SDS, linking effector recognition to biological function. The generality of stacking spines as conduits for effector-dependent, interdomain communication is discussed in light of their existence in adenine riboswitches, as well as the turnip yellow mosaic virus ribosome sensor.
Assuntos
Simulação de Dinâmica Molecular , Riboswitch , Pareamento de Bases , Regulação Bacteriana da Expressão Gênica , Guanina/análogos & derivados , Dodecilsulfato de Sódio/química , Thermoanaerobacter/genéticaRESUMO
Isothermal titration calorimetry (ITC) is a powerful biophysical tool to characterize energetic profiles of biomacromolecular interactions without any alteration of the underlying chemical structures. In this protocol, we describe procedures for performing, analyzing, and interpreting ITC data obtained from a cooperative riboswitch-ligand interaction.
Assuntos
Riboswitch , Calorimetria/métodos , Ligantes , Ligação Proteica , TermodinâmicaRESUMO
Riboswitches are structured non-coding RNAs often located upstream of essential genes in bacterial messenger RNAs. Such RNAs regulate expression of downstream genes by recognizing a specific cellular effector. Although nearly 50 riboswitch classes are known, only a handful recognize multiple effectors. Here, we report the 2.60-Å resolution co-crystal structure of a class I type I preQ1-sensing riboswitch that reveals two effectors stacked atop one another in a single binding pocket. These effectors bind with positive cooperativity in vitro and both molecules are necessary for gene regulation in bacterial cells. Stacked effector recognition appears to be a hallmark of the largest subgroup of preQ1 riboswitches, including those from pathogens such as Neisseria gonorrhoeae. We postulate that binding to stacked effectors arose in the RNA World to closely position two substrates for RNA-mediated catalysis. These findings expand known effector recognition capabilities of riboswitches and have implications for antimicrobial development.