Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3' end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields μmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA.

Here we show how fast dynamics between radicals and solvent molecules in liquid solutions can be detected by comparison of coupling factors determined by nuclear magnetic relaxation dispersion (NMRD) measurements and dynamic nuclear polarization (DNP) enhancement measurements at high magnetic field (9.2 T). This is important for a theoretical understanding of the Overhauser DNP mechanism at high magnetic fields and thus for optimization of the DNP agent/target system for high resolution liquid state NMR applications. Mixtures of the solution of TEMPOL radicals in water, toluene, acetone and DMSO have been investigated. The results are compared to the classical hard-sphere model and molecular dynamic simulations. Our results clearly indicate that fast sub-ps dynamics, which are not related to classical rotational or translational motion of the molecules, significantly contribute to the Overhauser DNP mechanism at high magnetic fields.

In bacteria, the regulation of gene expression by cis-acting transcriptional riboswitches located in the 5'-untranslated regions of messenger RNA requires the temporal synchronization of RNA synthesis and ligand binding-dependent conformational refolding. Ligand binding to the aptamer domain of the riboswitch induces premature termination of the mRNA synthesis of ligand-associated genes due to the coupled formation of 3'-structural elements acting as terminators. To date, there has been no high resolution structural description of the concerted process of synthesis and ligand-induced restructuring of the regulatory RNA element. Here, we show that for the guanine-sensing xpt-pbuX riboswitch from Bacillus subtilis, the conformation of the full-length transcripts is static: it exclusively populates the functional off-state but cannot switch to the on-state, regardless of the presence or absence of ligand. We show that only the combined matching of transcription rates and ligand binding enables transcription intermediates to undergo ligand-dependent conformational refolding.DOI: http://dx.doi.org/10.7554/eLife.21297.001

Gene repression induced by the formation of transcriptional terminators represents a prime example for the coupling of RNA synthesis, folding, and regulation. In this context, mapping the changes in available conformational space of transcription intermediates during RNA synthesis is important to understand riboswitch function. A majority of riboswitches, an important class of small metabolite-sensing regulatory RNAs, act as transcriptional regulators, but the dependence of ligand binding and the subsequent allosteric conformational switch on mRNA transcript length has not yet been investigated. We show a strict fine-tuning of binding and sequence-dependent alterations of conformational space by structural analysis of all relevant transcription intermediates at single-nucleotide resolution for the I-A type 2'dG-sensing riboswitch from Mesoplasma florum by NMR spectroscopy. Our results provide a general framework to dissect the coupling of synthesis and folding essential for riboswitch function, revealing the importance of metastable states for RNA-based gene regulation.

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.

No abstract

In RNA secondary structure determination, it is essential to determine whether a nucleotide is base-paired and not. Base-pairing of nucleotides is mediated by hydrogen bonds. The NMR characterization of hydrogen bonds relies on experiments correlating the NMR resonances of exchangeable protons and can be best performed for structured parts of the RNA, where labile hydrogen atoms are protected from solvent exchange. Functionally important regions in RNA, however, frequently reveal increased dynamic disorder which often leads to NMR signals of exchangeable protons that are broadened beyond (1)H detection. Here, we develop (13)C direct detected experiments to observe all nucleotides in RNA irrespective of whether they are involved in hydrogen bonds or not. Exploiting the self-decoupling of scalar couplings due to the exchange process, the hydrogen bonding behavior of the hydrogen bond donor of each individual nucleotide can be determined. Furthermore, the adaption of HNN-COSY experiments for (13)C direct detection allows correlations of donor-acceptor pairs and the localization of hydrogen-bond acceptor nucleotides. The proposed (13)C direct detected experiments therefore provide information about molecular sites not amenable by conventional proton-detected methods. Such information makes the RNA secondary structure determination by NMR more accurate and helps to validate secondary structure predictions based on bioinformatics.

Paramagnetic relaxation enhancement (PRE) is a versatile tool for NMR spectroscopic structural and kinetic studies in biological macromolecules. Here, we compare the quality of PRE data derived from two spin labels with markedly different dynamic properties for large RNAs using the I-A riboswitch aptamer domain (78 nt) from Mesoplamsa florum as model system. We designed two I-A aptamer constructs that were spin-labeled by noncovalent hybridization of short spin-labeled oligomer fragments. As an example of a flexible spin label, U-TEMPO was incorporated into the 3' terminal end of helix P1 while, the recently developed rigid spin-label Çm was incorporated in the 5' terminal end of helix P1. We determined PRE rates obtained from aromaticC bound proton intensities and compared these rates to PREs derived from imino proton intensities in this sizeable RNA (~78 nt). PRE restraints derived from both imino and aromatic protons yielded similar data quality, and hence can both be reliably used for PRE determination. For NMR, the data quality derived from the rigid spin label Çm is slightly better than the data quality for the flexible TEMPO as judged by comparison of the structural agreement with the I-A aptamer crystal structure (3SKI).