Structure of an internal loop motif with three consecutive U•U mismatches from stem-loop 1 in the 3'-UTR of the SARS-CoV-2 genomic RNA.

The single-stranded RNA genome of SARS-CoV-2 is highly structured. Numerous helical stem-loop structures interrupted by mismatch motifs are present in the functionally important 5'- and 3'-UTRs. These mismatches modulate local helical geometries and feature unusual arrays of hydrogen bonding donor and acceptor groups. However, their conformational and dynamical properties cannot be directly inferred from chemical probing and are difficult to predict theoretically. A mismatch motif (SL1-motif) consisting of three consecutive U•U base pairs is located in stem-loop 1 of the 3'-UTR. We combined NMR-spectroscopy and MD-simulations to investigate its structure and dynamics. All three U•U base pairs feature two direct hydrogen bonds and are as stable as Watson-Crick A:U base pairs. Plasmodium falciparum 25S rRNA contains a triple U•U mismatch motif (Pf-motif) differing from SL1-motif only with respect to the orientation of the two closing base pairs. Interestingly, while the geometry of the outer two U•U mismatches was identical in both motifs the preferred orientation of the central U•U mismatch was different. MD simulations and potassium ion titrations revealed that the potassium ion-binding mode to the major groove is connected to the different preferred geometries of the central base pair in the two motifs.

The 5'-terminal stem-loop RNA element of SARS-CoV-2 features highly dynamic structural elements that are sensitive to differences in cellular pH.

We present the nuclear magnetic resonance spectroscopy (NMR) solution structure of the 5'-terminal stem loop 5_SL1 (SL1) of the SARS-CoV-2 genome. SL1 contains two A-form helical elements and two regions with non-canonical structure, namely an apical pyrimidine-rich loop and an asymmetric internal loop with one and two nucleotides at the 5'- and 3'-terminal part of the sequence, respectively. The conformational ensemble representing the averaged solution structure of SL1 was validated using NMR residual dipolar coupling (RDC) and small-angle X-ray scattering (SAXS) data. We show that the internal loop is the major binding site for fragments of low molecular weight. This internal loop of SL1 can be stabilized by an A12-C28 interaction that promotes the transient formation of an A+•C base pair. As a consequence, the pKa of the internal loop adenosine A12 is shifted to 5.8, compared to a pKa of 3.63 of free adenosine. Furthermore, applying a recently developed pH-differential mutational profiling (PD-MaP) approach, we not only recapitulated our NMR findings of SL1 but also unveiled multiple sites potentially sensitive to pH across the 5'-UTR of SARS-CoV-2.

Structural and dynamic effects of pseudouridine modifications on noncanonical interactions in RNA.

Pseudouridine is the most frequently naturally occurring RNA modification, found in all classes of biologically functional RNAs. Compared to uridine, pseudouridine contains an additional hydrogen bond donor group and is therefore widely regarded as a structure stabilizing modification. However, the effects of pseudouridine modifications on the structure and dynamics of RNAs have so far only been investigated in a limited number of different structural contexts. Here, we introduced pseudouridine modifications into the U-turn motif and the adjacent U:U closing base pair of the neomycin-sensing riboswitch (NSR)-an extensively characterized model system for RNA structure, ligand binding, and dynamics. We show that the effects of replacing specific uridines with pseudouridines on RNA dynamics crucially depend on the exact location of the replacement site and can range from destabilizing to locally or even globally stabilizing. By using a combination of NMR spectroscopy, MD simulations and QM calculations, we rationalize the observed effects on a structural and dynamical level. Our results will help to better understand and predict the consequences of pseudouridine modifications on the structure and function of biologically important RNAs.

Development of a novel tobramycin dependent riboswitch.

We herein report the selection and characterization of a new riboswitch dependent on the aminoglycoside tobramycin. Its dynamic range rivals even the tetracycline dependent riboswitch to be the current best performing, synthetic riboswitch that controls translation initiation. The riboswitch was selected with RNA Capture-SELEX, a method that not only selects for binding but also for structural changes in aptamers on binding. This study demonstrates how this method can fundamentally reduce the labour required for the de novo identification of synthetic riboswitches. The initially selected riboswitch candidate harbours two distinct tobramycin binding sites with KDs of 1.1 nM and 2.4 µM, respectively, and can distinguish between tobramycin and the closely related compounds kanamycin A and B. Using detailed genetic and biochemical analyses and 1H NMR spectroscopy, the proposed secondary structure of the riboswitch was verified and the tobramycin binding sites were characterized. The two binding sites were found to be essentially non-overlapping, allowing for a separate investigation of their contribution to the activity of the riboswitch. We thereby found that only the high-affinity binding site was responsible for regulatory activity, which allowed us to engineer a riboswitch from only this site with a minimal sequence size of 33 nt and outstanding performance.

High-resolution structure of stem-loop 4 from the 5'-UTR of SARS-CoV-2 solved by solution state NMR.

We present the high-resolution structure of stem-loop 4 of the 5'-untranslated region (5_SL4) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) genome solved by solution state nuclear magnetic resonance spectroscopy. 5_SL4 adopts an extended rod-like structure with a single flexible looped-out nucleotide and two mixed tandem mismatches, each composed of a G•U wobble base pair and a pyrimidine•pyrimidine mismatch, which are incorporated into the stem-loop structure. Both the tandem mismatches and the looped-out residue destabilize the stem-loop structure locally. Their distribution along the 5_SL4 stem-loop suggests a role of these non-canonical elements in retaining functionally important structural plasticity in particular with regard to the accessibility of the start codon of an upstream open reading frame located in the RNA's apical loop. The apical loop-although mostly flexible-harbors residual structural features suggesting an additional role in molecular recognition processes. 5_SL4 is highly conserved among the different variants of SARS-CoV-2 and can be targeted by small molecule ligands, which it binds with intermediate affinity in the vicinity of the non-canonical elements within the stem-loop structure.

Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by 19 F NMR.

The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of 19 F NMR methods to characterize complex exchange processes with multiple excited states.

The COVID19-NMR Consortium: A Public Report on the Impact of this New Global Collaboration.

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.

Structure of an RNA aptamer in complex with the fluorophore tetramethylrhodamine.

RNA aptamers-artificially created RNAs with high affinity and selectivity for their target ligand generated from random sequence pools-are versatile tools in the fields of biotechnology and medicine. On a more fundamental level, they also further our general understanding of RNA-ligand interactions e. g. in regard to the relationship between structural complexity and ligand affinity and specificity, RNA structure and RNA folding. Detailed structural knowledge on a wide range of aptamer-ligand complexes is required to further our understanding of RNA-ligand interactions. Here, we present the atomic resolution structure of an RNA-aptamer binding to the fluorescent xanthene dye tetramethylrhodamine. The high resolution structure, solved by NMR-spectroscopy in solution, reveals binding features both common and different from the binding mode of other aptamers with affinity for ligands carrying planar aromatic ring systems such as the malachite green aptamer which binds to the tetramethylrhodamine related dye malachite green or the flavin mononucleotide aptamer.

Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy.

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 structure of the SAM/SAH-binding riboswitch.

S-adenosylmethionine (SAM) is a central metabolite since it is used as a methyl group donor in many different biochemical reactions. Many bacteria control intracellular SAM concentrations using riboswitch-based mechanisms. A number of structurally different riboswitch families specifically bind to SAM and mainly regulate the transcription or the translation of SAM-biosynthetic enzymes. In addition, a highly specific riboswitch class recognizes S-adenosylhomocysteine (SAH)-the product of SAM-dependent methyl group transfer reactions-and regulates enzymes responsible for SAH hydrolysis. High-resolution structures are available for many of these riboswitch classes and illustrate how they discriminate between the two structurally similar ligands SAM and SAH. The so-called SAM/SAH riboswitch class binds both ligands with similar affinities and is structurally not yet characterized. Here, we present a high-resolution nuclear magnetic resonance structure of a member of the SAM/SAH-riboswitch class in complex with SAH. Ligand binding induces pseudoknot formation and sequestration of the ribosome binding site. Thus, the SAM/SAH-riboswitches are translational 'OFF'-switches. Our results establish a structural basis for the unusual bispecificity of this riboswitch class. In conjunction with genomic data our structure suggests that the SAM/SAH-riboswitches might be an evolutionary late invention and not a remnant of a primordial RNA-world as suggested for other riboswitches.

Cooperation between a T†Domain and a Minimal C-Terminal Docking Domain to Enable Specific Assembly in a Multiprotein NRPS.

Non-ribosomal peptide synthetases (NRPS) produce natural products from amino acid building blocks. They often consist of multiple polypeptide chains which assemble in a specific linear order via specialized N- and C-terminal docking domains (N/C DDs). Typically, docking domains function independently from other domains in NRPS assembly. Thus, docking domain replacements enable the assembly of "designer" NRPS from proteins that normally do not interact. The multiprotein "peptide-antimicrobial-Xenorhabdus" (PAX) peptide-producing PaxS NRPS is assembled from the three proteins PaxA, PaxB and PaxC. Herein, we show that the small C DD of PaxA cooperates with its preceding thiolation (T1 ) domain to bind the N DD of PaxB with very high affinity, establishing a structural and thermodynamical basis for this unprecedented docking interaction, and we test its functional importance in vivo in a truncated PaxS assembly line. Similar docking interactions are apparently present in other NRPS systems.

An intricate balance of hydrogen bonding, ion atmosphere and dynamics facilitates a seamless uracil to cytosine substitution in the U-turn of the neomycin-sensing riboswitch.

The neomycin sensing riboswitch is the smallest biologically functional RNA riboswitch, forming a hairpin capped with a U-turn loop-a well-known RNA motif containing a conserved uracil. It was shown previously that a U→C substitution of the eponymous conserved uracil does not alter the riboswitch structure due to C protonation at N3. Furthermore, cytosine is evolutionary permitted to replace uracil in other U-turns. Here, we use molecular dynamics simulations to study the molecular basis of this substitution in the neomycin sensing riboswitch and show that a structure-stabilizing monovalent cation-binding site in the wild-type RNA is the main reason for its negligible structural effect. We then use NMR spectroscopy to confirm the existence of this cation-binding site and to demonstrate its effects on RNA stability. Lastly, using quantum chemical calculations, we show that the cation-binding site is altering the electronic environment of the wild-type U-turn so that it is more similar to the cytosine mutant. The study reveals an amazingly complex and delicate interplay between various energy contributions shaping up the 3D structure and evolution of nucleic acids.

Small but large enough: structural properties of armless mitochondrial tRNAs from the nematode Romanomermis culicivorax.

As adapter molecules to convert the nucleic acid information into the amino acid sequence, tRNAs play a central role in protein synthesis. To fulfill this function in a reliable way, tRNAs exhibit highly conserved structural features common in all organisms and in all cellular compartments active in translation. However, in mitochondria of metazoans, certain dramatic deviations from the consensus tRNA structure are described, where some tRNAs lack the D- or T-arm without losing their function. In Enoplea, this miniaturization comes to an extreme, and functional mitochondrial tRNAs can lack both arms, leading to a considerable size reduction. Here, we investigate the secondary and tertiary structure of two such armless tRNAs from Romanomermis culicivorax. Despite their high AU content, the transcripts fold into a single and surprisingly stable hairpin structure, deviating from standard tRNAs. The three-dimensional form is boomerang-like and diverges from the standard L-shape. These results indicate that such unconventional miniaturized tRNAs can still fold into a tRNA-like shape, although their length and secondary structure are very unusual. They highlight the remarkable flexibility of the protein synthesis apparatus and suggest that the translational machinery of Enoplea mitochondria may show compensatory adaptations to accommodate these armless tRNAs for efficient translation.

NMR experiments for the rapid identification of P=O···H-X type hydrogen bonds in nucleic acids.

Hydrogen bonds involving the backbone phosphate groups occur with high frequency in functional RNA molecules. They are often found in well-characterized tertiary structural motifs presenting powerful probes for the rapid identification of these motifs for structure elucidation purposes. We have shown recently that stable hydrogen bonds to the phosphate backbone can in principle be detected by relatively simple NMR-experiments, providing the identity of both the donor hydrogen and the acceptor phosphorous within the same experiment (Duchardt-Ferner et al., Angew Chem Int Ed Engl 50:7927-7930, 2011). However, for imino and hydroxyl hydrogen bond donor groups rapidly exchanging with the solvent as well as amino groups broadened by conformational exchange experimental sensitivity is severely hampered by extensive line broadening. Here, we present improved methods for the rapid identification of hydrogen bonds to phosphate groups in nucleic acids by NMR. The introduction of the SOFAST technique into 1H,31P-correlation experiments as well as a BEST-HNP experiment exploiting 3hJN,P rather than 2hJH,P coupling constants enables the rapid and sensitive identification of these hydrogen bonds in RNA. The experiments are applicable for larger RNAs (up to ~ 100-nt), for donor groups influenced by conformational exchange processes such as amino groups and for hydrogen bonds with rather labile hydrogens such as 2'-OH groups as well as for moderate sample concentrations. Interestingly, the size of the through-hydrogen bond scalar coupling constants depends not only on the type of the donor group but also on the structural context. The largest coupling constants were measured for hydrogen bonds involving the imino groups of protonated cytosine nucleotides as donors.

Evaluation of 15N-detected H-N correlation experiments on increasingly large RNAs.

Recently, 15N-detected multidimensional NMR experiments have been introduced for the investigation of proteins. Utilization of the slow transverse relaxation of nitrogen nuclei in a 15N-TROSY experiment allowed recording of high quality spectra for high molecular weight proteins, even in the absence of deuteration. Here, we demonstrate the applicability of three 15N-detected H-N correlation experiments (TROSY, BEST-TROSY and HSQC) to RNA. With the newly established 15N-detected BEST-TROSY experiment, which proves to be the most sensitive 15N-detected H-N correlation experiment, spectra for five RNA molecules ranging in size from 5 to 100 kDa were recorded. These spectra yielded high resolution in the 15N-dimension even for larger RNAs since the increase in line width with molecular weight is more pronounced in the 1H- than in the 15N-dimension. Further, we could experimentally validate the difference in relaxation behavior of imino groups in AU and GC base pairs. Additionally, we showed that 15N-detected experiments theoretically should benefit from sensitivity and resolution advantages at higher static fields but that the latter is obscured by exchange dynamics within the RNAs.

A Stably Protonated Adenine Nucleotide with a Highly Shifted pKa Value Stabilizes the Tertiary Structure of a GTP-Binding RNA Aptamer.

RNA tertiary structure motifs are stabilized by a wide variety of hydrogen-bonding interactions. Protonated A and C nucleotides are normally not considered to be suitable building blocks for such motifs since their pKa values are far from physiological pH. Here, we report the NMR solution structure of an in vitro selected GTP-binding RNA aptamer bound to GTP with an intricate tertiary structure. It contains a novel kind of base quartet stabilized by a protonated A residue. Owing to its unique structural environment in the base quartet, the pKa value for the protonation of this A residue in the complex is shifted by more than 5 pH units compared to the pKa for A nucleotides in single-stranded RNA. This is the largest pKa shift for an A residue in structured nucleic acids reported so far, and similar in size to the largest pKa shifts observed for amino acid side chains in proteins. Both RNA pre-folding and ligand binding contribute to the pKa shift.

The Solution Structure of the Lantibiotic Immunity Protein NisI and Its Interactions with Nisin.

Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity.

Building a stable RNA U-turn with a protonated cytidine.

The U-turn is a classical three-dimensional RNA folding motif first identified in the anticodon and T-loops of tRNAs. It also occurs frequently as a building block in other functional RNA structures in many different sequence and structural contexts. U-turns induce sharp changes in the direction of the RNA backbone and often conform to the 3-nt consensus sequence 5'-UNR-3' (N = any nucleotide, R = purine). The canonical U-turn motif is stabilized by a hydrogen bond between the N3 imino group of the U residue and the 3' phosphate group of the R residue as well as a hydrogen bond between the 2'-hydroxyl group of the uridine and the N7 nitrogen of the R residue. Here, we demonstrate that a protonated cytidine can functionally and structurally replace the uridine at the first position of the canonical U-turn motif in the apical loop of the neomycin riboswitch. Using NMR spectroscopy, we directly show that the N3 imino group of the protonated cytidine forms a hydrogen bond with the backbone phosphate 3' from the third nucleotide of the U-turn analogously to the imino group of the uridine in the canonical motif. In addition, we compare the stability of the hydrogen bonds in the mutant U-turn motif to the wild type and describe the NMR signature of the C+-phosphate interaction. Our results have implications for the prediction of RNA structural motifs and suggest simple approaches for the experimental identification of hydrogen bonds between protonated C-imino groups and the phosphate backbone.

What a Difference an OH Makes: Conformational Dynamics as the Basis for the Ligand Specificity of the Neomycin-Sensing Riboswitch.

To ensure appropriate metabolic regulation, riboswitches must discriminate efficiently between their target ligands and chemically similar molecules that are also present in the cell. A remarkable example of efficient ligand discrimination is a synthetic neomycin-sensing riboswitch. Paromomycin, which differs from neomycin only by the substitution of a single amino group with a hydroxy group, also binds but does not flip the riboswitch. Interestingly, the solution structures of the two riboswitch-ligand complexes are virtually identical. In this work, we demonstrate that the local loss of key intermolecular interactions at the substitution site is translated through a defined network of intramolecular interactions into global changes in RNA conformational dynamics. The remarkable specificity of this riboswitch is thus based on structural dynamics rather than static structural differences. In this respect, the neomycin riboswitch is a model for many of its natural counterparts.