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Up to now, NMR spectroscopic investigations of RNA have utilized imino proton resonances as reporters for base pairing and RNA structure. The nucleobase amino groups are often neglected, since most of their resonances are broadened beyond detection due to rotational motion around the C-NH2 bond. Here, we present 13 C-detected NMR experiments for the characterization of all RNA amino groups irrespective of their motional behavior. We have developed a C(N)H-HDQC experiment that enables the observation of a complete set of sharp amino resonances through the detection of proton-NH2 double quantum coherences. Further, we present an "amino"-NOESY experiment to detect NOEs to amino protons, which are undetectable by any other conventional NOESY experiment. Together, these experiments allow the exploration of additional chemical shift information and inter-residual proton distances important for high-resolution RNA secondary and tertiary structure determination.

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.

RNA-Tertiarstrukturmotive werden durch viele verschiedene Wasserstoffbrucken stabilisiert. Protonierte A- und C-Nukleotide werden normalerweise nicht als geeignete Bausteine fur solche Motive betrachtet, da ihre pKa-Werte weit vom physiologischen pH-Wert entfernt sind. Hier beschreiben wir die NMR-Struktur eines in vitro selektierten GTP-bindenden RNA-Aptamers gebunden an GTP mit einer sehr komplexen Tertiarstruktur. Es enthalt ein neuartiges, durch ein protoniertes A stabilisiertes Basenquartett. Aufgrund der besonderen strukturellen Umgebung innerhalb des Basenquartetts im GTP-Komplex ist der pKa-Wert fur die Protonierung dieses A-Nukleotides im Vergleich zum pKa-Wert fur A-Nukleotide in einzelstrangiger RNA um mehr als funf pH-Einheiten verschoben. Dies ist die groste bisher beschriebene Verschiebung des pKa-Wertes fur ein A in strukturierten Nukleinsauren. Sowohl die Vorfaltung der RNA als auch die Ligandenbindung tragen zu dieser Verschiebung des pKa-Wertes bei.

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.

Synthetic riboswitches can serve as sophisticated genetic control devices in synthetic biology, regulating gene expression through direct RNA-ligand interactions. We analyzed a synthetic neomycin riboswitch, which folds into a stem loop structure with an internal loop important for ligand binding and regulation. It is closed by a terminal hexaloop containing a U-turn and a looped-out adenine. We investigated the relationship between sequence, structure, and biological activity in the terminal loop by saturating mutagenesis, ITC, and NMR. Mutants corresponding to the canonical U-turn fold retained biological activity. An improvement of stacking interactions in the U-turn led to an RNA element with slightly enhanced regulatory activity. For the first position of the U-turn motif and the looped out base, sequence-activity relationships that could not initially be explained on the basis of the structure of the aptamer-ligand complex were observed. However, NMR studies of these mutants revealed subtle relationships between structure and dynamics of the aptamer in its free or bound state and biological activity.

RNA-Schalter erfullen wichtige Funktionen in der Genregulation. Sie bestehen aus einer Aptamerdomane, die eine spezifische und hochaffine Bindung mit einem Liganden eingeht, und einer Expressionsplattform, die durch eine allosterische Umorientierung auf den Ligandbindungszustand der Aptamerdomane reagiert. Die Genexpression kann sowohl wahrend der Transkription wie auch wahrend der Translation reguliert werden, auserdem wurde postuliert, dass RNA-Schalter die Prozessierung von mRNA steuern. Dieser Kurzaufsatz fasst den Kenntnisstand uber die Strukturen und Regulationsmechanismen von RNA-Schaltern zusammen. Besonderes Augenmerk gilt sekundaren und tertiaren Wechselwirkungen, die die Faltung der globularen RNA-Struktur stabilisieren und so die Funktion der Aptamerdomanen bestimmen.

Riboswitches are highly structured RNA elements that control gene expression by binding directly to small metabolite molecules. Remarkably, many of these metabolites contain negatively charged phosphate groups that contribute significantly to the binding affinity. An example is the thiamine pyrophosphate-sensing riboswitch in the 5'-untranslated region of the E. coli thiM mRNA. This riboswitch binds, in order of decreasing affinity, to thiamine pyrophosphate (TPP), thiamine monophosphate (TMP), and thiamine, which contain two, one, and no phosphate groups, respectively. We examined the binding of TPP and TMP to this riboswitch by using (31)P NMR spectroscopy. Chemical-shift changes were observed for the alpha- and beta-phosphate group of TPP and the phosphate group of TMP upon RNA binding; this indicates that they are in close contact with the RNA. Titration experiments with paramagnetic Mn(2+) ions revealed strong line-broadening effects for both (31)P signals of the bound TPP; this indicates a Mg(2+) binding site in close proximity and suggests that the phosphate group(s) of the ligand is/are recognized in a magnesium ion-mediated manner by the RNA.