Translational riboswitches located in the 5' UTR of the messenger RNA (mRNA) regulate translation through variation of the accessibility of the ribosome binding site (RBS). These are the result of conformational changes in the riboswitch RNA governed by ligand binding. Here, we use a combination of single-molecule colocalization techniques (Single-Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) and Single-Molecule Kinetic Analysis of Ribosome Binding (SiM-KARB)) and microscale thermophoresis (MST) to investigate the adenine-sensing riboswitch in Vibrio vulnificus, focusing on the changes of accessibility between the ligand-free and ligand-bound states. We show that both methods faithfully report on the accessibility of the RBS within the riboswitch and that both methods identify an increase in accessibility upon adenine binding. Expanding on the regulatory context, we show the impact of the ribosomal protein S1 on the unwinding of the RNA secondary structure, thereby favoring ribosome binding even for the apo state. The determined rate constants suggest that binding of the ribosome is faster than the time required to change from the ON state to the OFF state, a prerequisite for efficient regulation decision.

The outbreak of COVID-19 in December 2019 required the formation of international consortia for a coordinated scientific effort to understand and combat the virus. In this Viewpoint Article, we discuss how the NMR community has gathered to investigate the genome and proteome of SARS-CoV-2 and tested them for binding to low-molecular-weight binders. External factors including extended lockdowns due to the global pandemic character of the viral infection triggered the transition from locally focused collaborative research conducted within individual research groups to digital exchange formats for immediate discussion of unpublished results and data analysis, sample sharing, and coordinated research between more than 50 groups from 18 countries simultaneously. We discuss key lessons that might pertain after the end of the pandemic and challenges that we need to address.

The E. coli DEAD-Box helicase RhlB is responsible for ATP-dependent unwinding of structured mRNA to facilitate RNA degradation by the protein complex degradosome. The allosteric interaction with complex partner RNase E is necessary to stimulate both, RhlB's ATPase and RNA unwinding activity to levels comparable with other DEAD-Box helicases. However, the structural changes of the helicase RhlB induced by binding of RNase E have not been characterized and how those lead to increased reaction rates has remained unclear. We investigated the origin of this activation for RNA substrates with different topologies. Using NMR spectroscopy and an RNA centered approach, we could show that RNase E binding increases the affinity of RhlB towards a subset of RNA substrates, which leads to increased ATP turnover rates. Most strikingly, our studies revealed that in presence of RNase E (694-790) RhlB induces a conformational change in an RNA duplex with 5'- overhang even in absence of ATP, leading to partial duplex opening. Those results indicate a unique and novel activation mode of RhlB among DEAD-Box helicases, as ATP binding is thought to be an essential prerequisite for RNA unwinding.

Riboswitches are bacterial mRNA structure elements regulating either transcription or translation of downstream genes in response to high‐affinity binding of a low molecular weight ligand. Among this diverse group of RNA structures, the class‐I preQ1 sensing riboswitches (QSW) stand out since they are the smallest known natural riboswitches. The preQ1 sensing riboswitches combine ligand sensing and functional control within a single structural domain that adopts a pseudoknot conformation encapsulating both the cognate ligand and the ribosome binding site. preQ1 sensing riboswitches also occur in thermophilic bacteria. In these cases, their tertiary structures have to be stable even at temperatures above 60 °C to be functional at the organism's optimal growth temperatures. Despite the available high‐resolution structures of these riboswitches, it is not yet understood which tertiary interactions are primarily responsible for their exceptional temperature stability. Here, we show that an intricate three‐dimensional network of non‐canonical interactions involving various non‐neighboring nucleobases is the origin of the riboswitch's thermostability. An essential part of this network is a so far undetected stably protonated cytidine. It is characterized by an exceptional high pKA value of >9.7 and could be unambiguously identified through the application of modern heteronuclear detected NMR experiments. Thus, the presence or absence of a single proton can modulate the formation of an RNA tertiary structure and ligand binding capacity under extreme environmental conditions.

The aggregation of amyloid-beta 42 (A beta 42) is directly related to the pathogenesis of Alzheimer's disease. Here, we have investigated the early stages of the aggregation process, during which most of the cytotoxic species are formed. A beta 42 aggregation kinetics, characterized by the quantification of A beta 42 monomer consumption, were tracked by real-time solution NMR spectroscopy (RT-NMR) allowing the impact that low-molecular-weight (LMW) inhibitors and modulators exert on the aggregation process to be analysed. Distinct differences in the A beta 42 kinetic profiles were apparent and were further investigated kinetically and structurally by using thioflavin T (ThT) and transmission electron microscopy (TEM), respectively. LMW inhibitors were shown to have a differential impact on early-state aggregation. Insight provided here could direct future therapeutic design based on kinetic profiling of the process of fibril formation.

More than 170 post-transcriptional modifications of RNAs have currently been identified. Detailed biophysical investigations of these modifications have been limited since large RNAs containing these post-transcriptional modifications are difficult to produce. Further, adequate readout of spectroscopic fingerprints are important, necessitating additional labeling procedures beyond the naturally occurring RNA modifications. Here, we report the chemo-enzymatic synthesis of RNA modifications and several structurally similar fluorine-modified analogs further optimizing a recently developed methodology.[1] This chemo-enzymatic method allows synthesis of also large RNAs. We were able to incorporate 16 modified nucleotides and 6 19 F-labeled nucleotides. To showcase the applicability of such modified large RNAs, we incorporated a 19 F-labeled cytidine into the aptamer domain of the 2'dG sensing riboswitch (2'dG-sw) from Mesoplasma florum, enabling characterizing RNA fold, ligand binding and kinetics. Thanks to the large chemical shift dispersion of 19 F, we can detect conformational heterogeneity in the apo state of the riboswitch.

Both experimental and theoretical structure determinationsof RNAshave remained challenging due to the intrinsic dynamics of RNAs. Wereport here an integrated nuclear magnetic resonance/molecular dynamics(NMR/MD) structure determination approach to describe the dynamicstructure of the CUUG tetraloop. We show that the tetraloop undergoessubstantial dynamics, leading to averaging of the experimental data.These dynamics are particularly linked to the temperature-dependentpresence of a hydrogen bond within the tetraloop. Interpreting theNMR data by a single structure represents the low-temperature structurewell but fails to capture all conformational states occurring at ahigher temperature. We integrate MD simulations, starting from structuresof CUUG tetraloops within the Protein Data Bank, with an extensiveset of NMR data, and provide a structural ensemble that describesthe dynamic nature of the tetraloop and the experimental NMR datawell. We thus show that one of the most stable and frequently foundRNA tetraloops displays substantial dynamics, warranting such an integratedstructural approach.

AbstractDer Ausbruch von COVID‐19 im Dezember 2019 erforderte eine Koordination wissenschaftlicher Beiträge zum Verständnis und zur Bekämpfung des Virus und führte so zur Bildung internationaler Forschungskonsortien. In diesem Viewpoint Article erläutern wir, wie sich die NMR‐Community versammelt hat, um sowohl das Genom als auch das Proteom von SARS‐CoV‐2 strukturell zu untersuchen und für beide die Bindung an niedermolekulare Verbindungen zu testen. Externe Faktoren, einschließlich längerfristiger Lockdowns aufgrund der Pandemie, begünstigten den Übergang von lokaler, auf einzelne Forschungsgruppen ausgerichteter Verbundforschung hin zu hochkoordinierter Forschung mit über 50 Gruppen aus 18 Ländern, die durch digitale Austauschformate unmittelbar gemeinsam Daten analysierten und unveröffentlichte Ergebnisse diskutierten und darüber hinaus Proben untereinander teilten. Wir diskutieren hier einige wichtige Lehren, die nach dem Ende der Pandemie gezogen werden sollten, und Herausforderungen, die es anzugehen gilt.

The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.

Riboswitches are gene regulatory elements located in untranslated mRNA regions. They bind inducer molecules with high affinity and specificity. Cyclic-di-nucleotide-sensing riboswitches are major regulators of genes for the environment, membranes and motility (GEMM) of bacteria. Up to now, structural probing assays or crystal structures have provided insight into the interaction between cyclic-di-nucleotides and their corresponding riboswitches. ITC analysis, NMR analysis and computational modeling allowed us to gain a detailed understanding of the gene regulation mechanisms for the Cd1 (Clostridium difficile) and for the pilM (Geobacter metallireducens) riboswitches and their respective di-nucleotides c-di-GMP and c-GAMP. Binding capability showed a 25 nucleotide (nt) long window for pilM and a 61 nt window for Cd1. Within this window, binding affinities ranged from 35 μM to 0.25 μM spanning two orders of magnitude for Cd1 and pilM showing a strong dependence on competing riboswitch folds. Experimental results were incorporated into a Markov simulation to further our understanding of the transcriptional folding pathways of riboswitches. Our model showed the ability to predict riboswitch gene regulation and its dependence on transcription speed, pausing and ligand concentration.

A plethora of modified nucleotides extends the chemical and conformational space for natural occurring RNAs. tRNAs constitute the class of RNAs with the highest modification rate. The extensive modification modulates their overall stability, the fidelity and efficiency of translation. However, the impact of nucleotide modifications on the local structural dynamics is not well characterized. Here we show that the incorporation of the modified nucleotides in tRNAfMet from Escherichia coli leads to an increase in the local conformational dynamics, ultimately resulting in the stabilization of the overall tertiary structure. Through analysis of the local dynamics by NMR spectroscopic methods we find that, although the overall thermal stability of the tRNA is higher for the modified molecule, the conformational fluctuations on the local level are increased in comparison to an unmodified tRNA. In consequence, the melting of individual base pairs in the unmodified tRNA is determined by high entropic penalties compared to the modified. Further, we find that the modifications lead to a stabilization of long-range interactions harmonizing the stability of the tRNA's secondary and tertiary structure. Our results demonstrate that the increase in chemical space through introduction of modifications enables the population of otherwise inaccessible conformational substates.

Untranslated mRNA regions called riboswitches bind messenger molecules with high affinity and influence the expression of down stream genes. Cyclic-di-nucleotide sensing riboswitches are major regulators of lifestyle changes of bacteria. Current methodology is limited to indirect structural probing assays or the analysis of crystal structures inherently deprived of dynamics. A fast preparation method paired with ITC and NMR analysis of several transcriptional pilM intermediates allows a detailed understanding of this gene regulation mechanism.

Translational riboswitches are bacterial gene regulatory elements found in the 5'-untranslated region of mRNAs. They operate through a conformational refolding reaction that is triggered by a concentration change of a modulating small molecular ligand. The translation initiation region (TIR) is either released from or incorporated into base pairing interactions through the conformational switch. Hence, initiation of translation is regulated by the accessibility of the Shine-Dalgarno sequence and start codon. Interaction with the 30S ribosome is indispensable for the structural switch between functional OFF and ON states. However, on a molecular level it is still not fully resolved how the ribosome is accommodated near or at the translation initiation region in the context of translational riboswitches. The standby model of translation initiation postulates a binding site where the mRNA enters the ribosome and where it resides until the initiation site becomes unstructured and accessible. We here investigated the adenine-sensing riboswitch from Vibrio vulnificus. By application of a 19F labelling strategy for NMR spectroscopy that utilizes ligation techniques to synthesize differentially 19F labelled riboswitch molecules we show that nucleotides directly downstream of the riboswitch domain are first involved in productive interaction with the 30S ribosomal subunit. Upon the concerted action of ligand and the ribosomal protein rS1 the TIR becomes available and subsequently the 30S ribosome can slide towards the TIR. It will be interesting to see whether this is a general feature in translational riboswitches or if riboswitches exist where this region is structured and represent yet another layer of regulation.

SARS‐CoV‐2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti‐virals. Within the international Covid19‐NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR‐detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure‐based drug design against the SCoV2 proteome.

tRNAs are L-shaped RNA molecules of ~ 80 nucleotides that are responsible for decoding the mRNA and for the incorporation of the correct amino acid into the growing peptidyl-chain at the ribosome. They occur in all kingdoms of life and both their functions, and their structure are highly conserved. The L-shaped tertiary structure is based on a cloverleaf-like secondary structure that consists of four base paired stems connected by three to four loops. The anticodon base triplet, which is complementary to the sequence of the mRNA, resides in the anticodon loop whereas the amino acid is attached to the sequence CCA at the 3′-terminus of the molecule. tRNAs exhibit very stable secondary and tertiary structures and contain up to 10% modified nucleotides. However, their structure and function can also be maintained in the absence of nucleotide modifications. Here, we present the assignments of nucleobase resonances of the non-modified 77 nt tRNAIle from the gram-negative bacterium Escherichia coli. We obtained assignments for all imino resonances visible in the spectra of the tRNA as well as for additional exchangeable and non-exchangeable protons and for heteronuclei of the nucleobases. Based on these assignments we could determine the chemical shift differences between modified and non-modified tRNAIle as a first step towards the analysis of the effect of nucleotide modifications on tRNA’s structure and dynamics.

In bacteria RNA gene regulatory elements refold dependent on environmental clues between two or more long-lived conformational states each associated with a distinct regulatory state. The refolding kinetics are strongly temperature-dependent and especially at lower temperatures they reach timescales that are biologically not accessible. To overcome this problem, RNA chaperones have evolved. However, the precise molecular mechanism of how these proteins accelerate RNA refolding reactions remains enigmatic. Here we show how the RNA chaperone StpA of Escherichia coli leads to an acceleration of a bistable RNA’s refolding kinetics through the selective destabilization of key base pairing interactions. We find in laser assisted real-time NMR experiments on photocaged bistable RNAs that the RNA chaperone leads to a two-fold increase in refolding rates at low temperatures due to reduced stability of ground state conformations. Further, we can show that upon interaction with StpA, base pairing interactions in the bistable RNA are modulated to favor refolding through the dominant pseudoknotted transition pathway. Our results shed light on the molecular mechanism of the interaction between RNA chaperones and bistable RNAs and are the first step into a functional classification of chaperones dependent on their biophysical mode of operation.

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.

The review describes the application of nuclear magnetic resonance (NMR) spectroscopy to study kinetics of folding, refolding and aggregation of proteins, RNA and DNA. Time-resolved NMR experiments can be conducted in a reversible or an irreversible manner. In particular, irreversible folding experiments pose large requirements for (i) signal-to-noise due to the time limitations and (ii) synchronising of the refolding steps. Thus, this contribution discusses the application of methods for signal-to-noise increases, including dynamic nuclear polarisation, hyperpolarisation and photo-CIDNP for the study of time-resolved NMR studies. Further, methods are reviewed ranging from pressure and temperature jump, light induction to rapid mixing to induce rapidly non-equilibrium conditions required to initiate folding.

Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.

The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5′- and 3′-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5_SL4) is located within the 5′-untranslated region (5′-UTR) important for viral replication. 5_SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1H, 13C, 15N and 31P resonance assignments of 5_SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy.