We coupled the antimicrobial activity of two well-studied lactoferricin derivatives, LF11-215 and LF11-324, in Escherichia coli and different lipid-only mimics of its cytoplasmic membrane using a common thermodynamic framework for peptide partitioning. In particular, we combined an improved analysis of microdilution assays with ζ-potential measurements, which allowed us to discriminate between the maximum number of surface-adsorbed peptides and peptides fully partitioned into the bacteria. At the same time, we measured the partitioning of the peptides into vesicles composed of phosphatidylethanolamine (PE), phosphatidylgylcerol (PG), and cardiolipin (CL) mixtures using tryptophan fluorescence and determined their membrane activity using a dye leakage assay and small-angle X-ray scattering. We found that the vast majority of LF11-215 and LF11-324 readily enter inner bacterial compartments, whereas only 1−5% remain surface bound. We observed comparable membrane binding of both peptides in membrane mimics containing PE and different molar ratios of PG and CL. The peptides' activity caused a concentration-dependent dye leakage in all studied membrane mimics; however, it also led to the formation of large aggregates, part of which contained collapsed multibilayers with sandwiched peptides in the interstitial space between membranes. This effect was least pronounced in pure PG vesicles, requiring also the highest peptide concentration to induce membrane permeabilization. In PE-containing systems, we additionally observed an effective shielding of the fluorescent dyes from leakage even at highest peptide concentrations, suggesting a coupling of the peptide activity to vesicle fusion, being mediated by the intrinsic lipid curvatures of PE and CL. Our results thus show that LF11-215 and LF11-324 effectively target inner bacterial components, while the stored elastic stress makes membranes more vulnerable to peptide translocation.

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.

Using chemical synthesis and solution NMR spectroscopy, RNA structural ensembles including a major ground state and minor populated excited states can be studied at atomic resolution. In this work, atom-specific C labeled RNA building blocks - a 5-C-uridine and a 2,8-C-adenosine building block - are used to introduce isolated C-H-spin topologies into a target RNA to probe such structural ensembles via NMR spectroscopy. First, the 5-C-uridine 2'-O-TBDMS-phosphoramidite building block was introduced into a 21 nucleotide (nt) tP5c stem construct of the tP5abc subdomain of the Tetrahymena group I ribozyme. Then, the 2,8-C-adenosine 2'-O-TBDMS-phosphoramidite building block was incorporated into a 9 kDa and a 15 kD construct derived from the epsilon (ε) RNA element of the duck Hepatitis B virus. The 2,8-C-adenosine resonances of the 9 kDa 28 nt sequence could be mapped to the full-length 53 nt construct. The isolated NMR active nuclei pairs were used to probe for low populated excited states (<10%) via C-Carr-Purcell-Meiboom-Gill (CPMG)-relaxation dispersion NMR spectroscopy. TheC-CPMG relaxation dispersion experiment recapitulated a secondary structure switching event in the P5c hairpin of the group I intron construct previously revealed by N relaxation dispersion experiments. In the ε-HBV RNA an unfolding event occurring on the millisecond time scale was found in the upper stem in-line with earlier observations. This unpaired conformational state is presumed to be important for the binding of the epsilon reverse transcriptase (RT) enzyme. Thus, a full description of an RNA's folding landscape helps to obtain a deeper understanding of its function, as these high energy conformational states often represent functionally important intermediates involved in (un)folding or ribozyme catalysis.

The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. We found the hydrogen bond between the N1 of purines and the N3 of pyrimidines to be sufficient for decoding of the first two codon nucleotides, whereas adequate stacking between the RNA bases is critical at the wobble position. Inosine, found in eukaryotic mRNAs, is an important example of destabilization of the codon-anticodon interaction. Whereas single inosines are efficiently translated, multiple inosines, e.g., in the serotonin receptor 5-HT2C mRNA, inhibit translation. Thus, our results indicate that despite the robustness of the decoding process, its tolerance toward the weakening of codon-anticodon interactions is limited.

We report the synthesis of atom-specifically 13C-modified building blocks that can be incorporated into DNA via solid phase synthesis to facilitate investigations on structural and dynamic features via NMR spectroscopy. In detail, 6-13C-modified pyrimidine and 8-13C purine DNA phosphoramidites were synthesized and incorporated into a polypurine tract DNA/RNA hybrid duplex to showcase the facile resonance assignment using site-specific labeling. We also addressed micro- to millisecond dynamics in the mini-cTAR DNA. This DNA is involved in the HIV replication cycle and our data points toward an exchange process in the lower stem of the hairpin that is up-regulated in the presence of the HIV-1 nucleocapsid protein 7. As another example, we picked a G-quadruplex that was earlier shown to exist in two folds. Using site-specific 8-13C-2′deoxyguanosine labeling we were able to verify the slow exchange between the two forms on the chemical shift time scale. In a real-time NMR experiment the re-equilibration of the fold distribution after a T-jump could be monitored yielding a rate of 0.012 min−1. Finally, we used 13C-ZZ-exchange spectroscopy to characterize the kinetics between two stacked X-conformers of a Holliday junction mimic. At 25°C, the refolding process was found to occur at a forward rate constant of 3.1 s−1 and with a backward rate constant of 10.6 s−1.

We showcase the high potential of the 2 0 -cyanoethoxymethyl (CEM) methodology to synthesize RNAs with naturally occurring modified residues carrying stable isotope (SI) labels for NMR spectro- Solution and solid state nuclear magnetic resonance (NMR) spectroscopy have proven to be highly suitable to address structural and dynamic features of RNA. 1-4 A prerequisite to apply state-of-the-art NMR experiments is the introduction of a stable isotope (SI) labelling pattern using 13 C/ 15 N labelled RNA or DNA precursors. [5][6][7][8] The most wide-spread method uses labelled (2 0 -deoxy)-ribonucleotide triphosphates and enzymes to produce the desired RNA or DNA sequence enriched with 13 C and 15 N nuclei. 1,5 This approach enables to produce sufficient amounts of RNA and DNA for NMR spectroscopic applications. This well-established method allows nucleotide specific labeling by mixing a SI-labeled with unlabeled d/rNTPs. Especially in larger RNAs (460 nt) such nucleotide specific SI-labeling can still lead to significant resonance overlap. That is why, the PLOR (position-selective labelling of RNA) method was recently introduced, which holds the promise to site-specifically label RNA using SI-labelled ribonucleotide triphosphates and T7 RNA polymerase. 9 An alternative method was concurrently developed making use of the synthesis of 2 0 -O-tri-iso-propylsilyloxymethylphosphoramidites and solid phase synthesis. 10-13The approach works well for medium sized RNAs up to 50 nts and the synthetic access to the SI-labelled building blocks is well established.10,12 Thus, the fully chemical SI-labelling protocol can be regarded as an expedient expansion to the settled enzymatic procedures to freely chose the number and positioning of SI-labeled residues into a target RNA. In our hands, however, the standard solid phase synthesis methods are not that well suited to produce larger amounts (450 nmol) and purities higher than 95% for RNAs exceeding 60 nts. Due to this restriction, large RNAs are only accessible via enzymatic ligation strategies using T4 RNA/DNA ligase making extra optimization steps necessary or introducing new problems, such as finding the optimal ligation site or issues regarding up-scaling and yield of the ligation product. 14-16 Thus, an improved synthetic procedure to directly address SI-labelling of larger RNAs (460 nt) at amounts suitable for NMR would be highly desirable. We report the synthesis of SI-labelled RNAs ranging in size between 60 to 80 nts capitalizing on the 2 0 -cyanoethoxymethyl (CEM) RNA synthesis method. 17,18 As these CEM building blocks are not commercially available all phosphoramidites were produced in-house and we further synthesized 13 C-/ 15 N-labelled unmodified and naturally occurring modified RNA phosphoramidites (Fig. 1a and b). In detail, we focused on the synthesis of 8- We used these monomer units to produce SI-labelled RNAs exceeding the size limitation of 60 nucleotides for NMR up to 20 nucleotides. The RNAs reported here were synthesized on a 1.3 mmol scale and on a 1000...

Although group II intron ribozymes are intensively studied the question how structural dynamics affects splicing catalysis has remained elusive. We report for the first time that the group II intron domain 6 exists in a secondary structure equilibrium between a single- and a two-nucleotide bulge conformation, which is directly linked to a switch between sugar puckers of the branch site adenosine. Our study determined a functional sugar pucker equilibrium between the transesterification active C2′-endo conformation of the branch site adenosine in the 1nt bulge and an inactive C3′-endo state in the 2nt bulge fold, allowing the group II intron to switch its activity from the branching to the exon ligation step. Our detailed NMR spectroscopic investigation identified magnesium (II) ions and the branching reaction as regulators of the equilibrium populations. The tuneable secondary structure/sugar pucker equilibrium supports a conformational selection mechanism to up- and downregulate catalytically active and inactive states of the branch site adenosine to orchestrate the multi-step splicing process. The conformational dynamics of group II intron domain 6 is also proposed to be a key aspect for the directionality selection in reversible splicing.

Watson–Crick like G‐U mismatches with tautomeric Genol or Uenol bases can evade fidelity checkpoints and thereby contribute to translational errors. The 5‐oxyacetic acid uridine (cmo5U) modification is a base modification at the wobble position on tRNAs and is presumed to expand the decoding capability of tRNA at this position by forming Watson–Crick like cmo5Uenol‐G mismatches. A detailed investigation on the influence of the cmo5U modification on structural and dynamic features of RNA was carried out by using solution NMR spectroscopy and UV melting curve analysis. The introduction of a stable isotope labeled variant of the cmo5U modifier allowed the application of relaxation dispersion NMR to probe the potentially formed Watson–Crick like cmo5Uenol‐G base pair. Surprisingly, we find that at neutral pH, the modification promotes transient formation of anionic Watson–Crick like cmo5U−‐G, and not enolic base pairs. Our results suggest that recoding is mediated by an anionic Watson–Crick like species, as well as bring an interesting aspect of naturally occurring RNA modifications into focus—the fine tuning of nucleobase properties leading to modulation of the RNA structural landscape by adoption of alternative base pairing patterns.