RESUMEN
Structural changes in RNAs are an important contributor to controlling gene expression not only at the posttranscriptional stage but also during transcription. A subclass of riboswitches and RNA thermometers located in the 5' region of the primary transcript regulates the downstream functional unit - usually an ORF - through premature termination of transcription. Not only such elements occur naturally, but they are also attractive devices in synthetic biology. The possibility to design such riboswitches or RNA thermometers is thus of considerable practical interest. Since these functional RNA elements act already during transcription, it is important to model and understand the dynamics of folding and, in particular, the formation of intermediate structures concurrently with transcription. Cotranscriptional folding simulations are therefore an important step to verify the functionality of design constructs before conducting expensive and labor-intensive wet lab experiments. For RNAs, full-fledged molecular dynamics simulations are far beyond practical reach because of both the size of the molecules and the timescales of interest. Even at the simplified level of secondary structures, further approximations are necessary. The BarMap approach is based on representing the secondary structure landscape for each individual transcription step by a coarse-grained representation that only retains a small set of low-energy local minima and the energy barriers between them. The folding dynamics between two transcriptional elongation steps is modeled as a Markov process on this representation. Maps between pairs of consecutive coarse-grained landscapes make it possible to follow the folding process as it changes in response to transcription elongation. In its original implementation, the BarMap software provides a general framework to investigate RNA folding dynamics on temporally changing landscapes. It is, however, difficult to use in particular for specific scenarios such as cotranscriptional folding. To overcome this limitation, we developed the user-friendly BarMap-QA pipeline described in detail in this contribution. It is illustrated here by an elaborate example that emphasizes the careful monitoring of several quality measures. Using an iterative workflow, a reliable and complete kinetics simulation of a synthetic, transcription-regulating riboswitch is obtained using minimal computational resources. All programs and scripts used in this contribution are free software and available for download as a source distribution for Linux® or as a platform-independent Docker® image including support for Apple macOS® and Microsoft Windows®.
Asunto(s)
Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Pliegue del ARN , Transcripción Genética , Riboswitch/genética , ARN/química , ARN/genética , Programas InformáticosRESUMEN
The in silico prediction of non-coding and protein-coding genetic loci has received considerable attention in comparative genomics aiming in particular at the identification of properties of nucleotide sequences that are informative of their biological role in the cell. We present here a software framework for the alignment-based training, evaluation and application of machine learning models with user-defined parameters. Instead of focusing on the one-size-fits-all approach of pervasive in silico annotation pipelines, we offer a framework for the structured generation and evaluation of models based on arbitrary features and input data, focusing on stable and explainable results. Furthermore, we showcase the usage of our software package in a full-genome screen of Drosophila melanogaster and evaluate our results against the well-known but much less flexible program RNAz.
RESUMEN
Gene regulation in prokaryotes often depends on RNA elements such as riboswitches or RNA thermometers located in the 5' untranslated region of mRNA. Rearrangements of the RNA structure in response, e.g., to the binding of small molecules or ions control translational initiation or premature termination of transcription and thus mRNA expression. Such structural responses are amenable to computational modelling, making it possible to rationally design synthetic riboswitches for a given aptamer. Starting from an artificial aptamer, we construct the first synthetic transcriptional riboswitches that respond to the antibiotic neomycin. We show that the switching behaviour in vivo critically depends not only on the sequence of the riboswitch itself, but also on its sequence context. We therefore developed in silico methods to predict the impact of the context, making it possible to adapt the design and to rescue non-functional riboswitches. We furthermore analyse the influence of 5' hairpins with varying stability on neomycin riboswitch activity. Our data highlight the limitations of a simple plug-and-play approach in the design of complex genetic circuits and demonstrate that detailed computational models significantly simplify, improve, and automate the design of transcriptional circuits. Our design software is available under a free licence on GitHub (https://github.com/xileF1337/riboswitch_design).
Asunto(s)
Clonación Molecular/métodos , Biología Computacional/métodos , Neomicina/química , Riboswitch/genética , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/genética , Simulación por Computador , Regulación Bacteriana de la Expresión Génica , Genes Reporteros/genética , Neomicina/farmacología , Conformación de Ácido Nucleico , ARN Bacteriano/análisis , ARN Bacteriano/química , ARN Bacteriano/genética , Programas Informáticos , Biología SintéticaRESUMEN
This contribution sketches a work flow to design an RNA switch that is able to adapt two structural conformations in a ligand-dependent way. A well characterized RNA aptamer, i.e., knowing its Kd and adaptive structural features, is an essential ingredient of the described design process. We exemplify the principles using the well-known theophylline aptamer throughout this work. The aptamer in its ligand-binding competent structure represents one structural conformation of the switch while an alternative fold that disrupts the binding-competent structure forms the other conformation. To keep it simple we do not incorporate any regulatory mechanism to control transcription or translation. We elucidate a commonly used design process by explicitly dissecting and explaining the necessary steps in detail. We developed a novel objective function which specifies the mechanistics of this simple, ligand-triggered riboswitch and describe an extensive in silico analysis pipeline to evaluate important kinetic properties of the designed sequences. This protocol and the developed software can be easily extended or adapted to fit novel design scenarios and thus can serve as a template for future needs.
Asunto(s)
Aptámeros de Nucleótidos/síntesis química , Biología Computacional/métodos , Conformación de Ácido Nucleico , Riboswitch/genética , Aptámeros de Nucleótidos/genética , Biología Computacional/instrumentación , Cinética , Ligandos , Pliegue del ARN , Programas InformáticosRESUMEN
BACKGROUND: The binding of small ligands to RNA elements can cause substantial changes in the RNA structure. This constitutes an important, fast-acting mechanism of ligand-controlled transcriptional and translational gene regulation implemented by a wide variety of riboswitches. The associated refolding processes often cannot be explained by thermodynamic effects alone. Instead, they are governed by the kinetics of RNA folding. While the computational analysis of RNA folding can make use of well-established models of the thermodynamics of RNA structures formation, RNA-RNA interaction, and RNA-ligand interaction, kinetic effects pose fundamentally more challenging problems due to the enormous size of the conformation space. The analysis of the combined process of ligand binding and structure formation even for small RNAs is plagued by intractably large state spaces. Moreover, the interaction is concentration-dependent and thus is intrinsically non-linear. This precludes the direct transfer of the strategies previously used for the analysis of RNA folding kinetics. RESULTS: In our novel, computationally tractable approach to RNA-ligand kinetics, we overcome the two main difficulties by applying a gradient-based coarse graining to RNA-ligand systems and solving the process in a pseudo-first order approximation. The latter is well-justified for the most common case of ligand excess in RNA-ligand systems. We present the approach rigorously and discuss the parametrization of the model based on empirical data. The method supports the kinetic study of RNA-ligand systems, in particular at different ligand concentrations. As an example, we apply our approach to analyze the concentration dependence of the ligand response of the rationally designed, artificial theophylline riboswitch RS3. CONCLUSION: This work demonstrates the tractability of the computational analysis of RNA-ligand interaction. Naturally, the model will profit as more accurate measurements of folding and binding parameters become available. Due to this work, computational analysis is available to support tasks like the design of riboswitches; our analysis of RS3 suggests strong co-transcriptional effects for this riboswitch. The method used in this study is available online, cf. Section "Availability of data and materials".