Shine-Dalgarno Accessibility Governs Ribosome Binding to the Adenine Riboswitch.

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.

Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development.

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.

Binding of 30S Ribosome Induces Single-stranded Conformation Within and Downstream of the Expression Platform in a Translational Riboswitch.

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.

NMR assignment of non-modified tRNAIle from Escherichia coli.

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.

RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics.

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.

Switching at the ribosome: riboswitches need rProteins as modulators to regulate translation.

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.

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications.

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

19 F NMR-Based Fragment Screening for 14 Different Biologically Active RNAs and 10 DNA and Protein Counter-Screens.

We report here the nuclear magnetic resonance 19 F screening of 14 RNA targets with different secondary and tertiary structure to systematically assess the druggability of RNAs. Our RNA targets include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. Based on counter-screens against five DNAs and five proteins, we can show that RNA can be specifically targeted. To demonstrate the quality of the initial fragment library that has been designed for easy follow-up chemistry, we further show how to increase binding affinity from an initial fragment hit by chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low-micromolar binding affinity without losing binding specificity between two different terminator structures.

Effect of grape juice, red wine and resveratrol solution on antioxidant, anti-inflammatory, hepactic function and lipid profile in rats feds with high-fat diet.

The objective was to investigate the role of consumption of grape juice (GJ), red wine (RW) or resveratrol solution (RS) on rats treated with a high-fat diet (HFD). Among the drinks offered, GJ had lower content of polyphenols and trans-resveratrol. Nevertheless, GJ showed similar content of anthocyanin and antioxidant activity to RW, although higher than RS. In rats treated with HFD, consumption of GJ presented best antioxidant and anti-inflammatory activity, reducing glutathione peroxidase and interleukin-6 serum levels. In addition, GJ promoted better levels of cholesterol and liver markers. On the other hand, RW aggravated the oxidizing effect of HFD, increasing catalase activity and interleukin-6 level. Already, RS showed no benefit in animals. Thus, GJ minimized the effects of HFD on oxidative stress and inflammation beyond promoted better levels of lipid profile and liver biomarkers. However, consumption of RS showed no benefit and RW revealed a pro-oxidant effect, as did HFD. [Figure: see text].

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.

Electrochemical study and simultaneous voltammetric determination of contraceptives ethinylestradiol and cyproterone acetate using silver nanoparticles solid amalgam electrode and cationic surfactant.

In this work we propose the voltammetric analysis of contraceptive hormones ethinylestradiol (EE) and cyproterone acetate (CPA) using solid amalgam electrode fabricated with silver nanoparticles. To the best of our knowledge, this is the first report describing the simultaneous determination of these two compounds and also the first report of the use of amalgam electrode for analysis of EE and CPA. The voltammetric behavior of both substances was investigated by their reduction. An irreversible electrochemical process involving two protons and two electrons was found for each compound. The analytical assays were carried out using staircase voltammetry (SCV). Due to this, aiming to improve the analytical sensitivity, the cationic surfactant cetyltrimethylammonium bromide was also used. The instrumental and experimental parameters were studied and optimized to achieve the best conditions for the analysis. Under the optimum conditions, the voltammetric signals of EE and CPA showed dependence on the concentration range from 6.4 × 10-7 to 7.8 × 10-6 mol L-1 and from 1.0 × 10-6 to 1.0 × 10-5 mol L-1, respectively. The limits of detection obtained were 1.03 × 10-7 mol L-1 for EE and 2.99 × 10-7 mol L-1 for CPA. The analytical usefulness of the method was evaluated through its application on the simultaneous determination of EE and CPA in pharmaceutical formulations and urine samples. The two analytes were successfully quantified in these samples with good precision and the values found presented satisfactory concordance with the reference values, suggesting acceptable analytical efficiency for the approach described here.

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.