RESUMO
The adenine-sensing riboswitch from the Gram-negative bacterium Vibrio vulnificus is an RNA-based gene regulatory element that acts in response to both its cognate low-molecular weight ligand and temperature. The combined sensitivity to environmental temperature and ligand concentration is maintained by an equilibrium of three distinct conformations involving two ligand-free states and one ligand-bound state. The key structural element that undergoes refolding in the ligand-free states comprises a 35-nucleotide temperature response module. Here, we present the structural characterization of this temperature response module. We employ high-resolution NMR spectroscopy and photocaged RNAs as molecular probes to decipher the kinetic and thermodynamic framework of the secondary structure transition in the apo state of the riboswitch. We propose a model for the transition state adopted during the thermal refolding of the temperature response module that connects two mutually exclusive long-lived and stable conformational states. This transition state is characterized by a comparatively low free activation enthalpy. A pseudoknot conformation in the transition state, as commonly seen in RNA refolding, is therefore unlikely. More likely, the transition state of the adenine-sensing riboswitch temperature response module features a linear conformation.
Assuntos
Riboswitch/genética , Riboswitch/fisiologia , Vibrio vulnificus/química , Aclimatação , Aptâmeros de Nucleotídeos/metabolismo , Cinética , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação de Ácido Nucleico , Dobramento de RNA/fisiologia , RNA Bacteriano/química , Temperatura , Termodinâmica , Vibrio vulnificus/metabolismoRESUMO
The complex folding energy landscape of DNA G-quadruplexes leads to numerous conformations for this functionally important class of noncanonical DNA structures. A new layer of conformational heterogeneity comes from sequences with different numbers of G-nucleotides in each of the DNA G-strands that form the four-stranded G-quartet core. The mechanisms by which G-quadruplexes transition from one folded conformation to another are currently unknown. To address this question, we studied two different G-quadruplexes, selecting a single conformation by blocking hydrogen bonding with photolabile protection groups. Upon irradiation, the block can be released and the kinetics of re-equilibration to the native conformational equilibrium can be determined by time-resolved NMR. We compared the NMR-derived refolding kinetics with data derived from thermal hysteresis folding kinetic experiments and found excellent agreement. The outlined methodological approach allows separation of K+-induced G-quadruplex formation and subsequent refolding and provides key insight into rate-limiting steps of G-quadruplex conformational dynamics.
Assuntos
DNA/química , Quadruplex G , Conformação de Ácido Nucleico , Cinética , Ressonância Magnética Nuclear BiomolecularRESUMO
Photolabile protecting groups are widely used to trigger oligonucleotide activity. The ON/OFF-amplitude is a critical parameter. An experimental setup has been developed to identify protecting group derivatives with superior caging properties. Bulky rests are attached to the cage moiety via Cu-catalyzed azide-alkyne cycloaddition post-synthetically on DNA. Interestingly, the decrease in melting temperature upon introducing o-nitrobenzyl-caged (NPBY-) and diethylaminocoumarin-cages (DEACM-) in DNA duplexes reaches a limiting value. NMR spectroscopy was used to characterize individual base-pair stabilities and determine experimental structures of a selected number of photocaged DNA molecules. The experimental structures agree well with structures predicted by MD simulations. Combined, the structural data indicate that once a sterically demanding group is added to generate a tri-substituted carbon, the sterically less demanding cage moiety points towards the neighboring nucleoside and the bulkier substituents remain in the major groove.
Assuntos
DNA/química , Nucleosídeos/química , Alcinos/química , Azidas/química , Pareamento de Bases , Catálise , Cobre/química , Reação de Cicloadição , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , EstereoisomerismoRESUMO
Transcriptional riboswitches modulate downstream gene expression by a tight coupling of ligand-dependent RNA folding kinetics with the rate of transcription. RNA folding pathways leading to functional ON and OFF regulation involve the formation of metastable states within well-defined sequence intervals during transcription. The kinetic requirements for the formation and preservation of these metastable states in the context of transcription remain unresolved. Here, we reversibly trap the previously defined regulatory relevant metastable intermediate of the Mesoplasma florum 2'-deoxyguanosine (2'dG)-sensing riboswitch using a photocaging-ligation approach, and monitor folding to its native state by real-time NMR in both presence and absence of ligand. We further determine transcription rates for two different bacterial RNA polymerases. Our results reveal that the riboswitch functions only at transcription rates typical for bacterial polymerases (10-50 nt s-1) and that gene expression is modulated by 40-50% only, while subtle differences in folding rates guide population ratios within the structural ensemble to a specific regulatory outcome.
Assuntos
Riboswitch/genética , Transdução de Sinais/genética , Transcrição Gênica , Sequência de Bases , Simulação por Computador , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Genéticos , Conformação de Ácido NucleicoRESUMO
A new photoactivatable fluorophore based on a quinone-cyanine-7 (QCy7) scaffold was synthesized and spectroscopically characterized. The fluorophore exhibits fluorescence in the near-infrared spectral window and a large Stokes shift. Its application in super-resolution microscopy as an antibody bioconjugate is demonstrated.