Correlation of nucleotide base and sugar protons in a 15N-labeled HIV-1 RNA oligonucleotide by 1H-15N HSQC experiments

151

188

Riboswitches are highly structured elements in the 5′-untranslated regions (5′-UTRs) of messenger RNA that control gene expression by specifically binding to small metabolite molecules. They consist of an aptamer domain responsible for ligand binding and an expression platform. Ligand binding in the aptamer domain leads to conformational changes in the expression platform that result in transcription termination or abolish ribosome binding. The guanine riboswitch binds with high-specificity to guanine and hypoxanthine and is among the smallest riboswitches described so far. The X-ray-structure of its aptamer domain in complex with guanine/hypoxanthine reveals an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base pairing interactions. We analyzed the conformational transitions of the aptamer domain induced by binding of hypoxanthine using high-resolution NMR-spectroscopy in solution. We found that the long-range base pairing interactions are already present in the free RNA and preorganize its global fold. The ligand binding core region is lacking hydrogen bonding interactions and therefore likely to be unstructured in the absence of ligand. Mg2+-ions are not essential for ligand binding and do not change the structure of the RNA-ligand complex but stabilize the structure at elevated temperatures. We identified a mutant RNA where the long-range base pairing interactions are disrupted in the free form of the RNA but form upon ligand binding in an Mg2+-dependent fashion. The tertiary interaction motif is stable outside the riboswitch context.

Section: Methodsmentioning

confidence: 99%

Interplay of 'induced fit' and preorganization in the ligand induced folding of the aptamer domain of the guanine binding riboswitch

Noeske

Buck²,

Fürtig³

et al. 2006

151

188

“…Water suppression was achieved using the WATERGATE water suppression scheme (22) including water flip back pulses (23). 1 H, 15 N-HSQC, 2 J HN - 1 H, 15 N-HSQC (24) and 2D- or 15 N-edited 3D-NOESY experiments were performed using standard pulse sequences (25). The 2h J NN -HNN-COSY experiments (26) were performed as described previously (15).…”

Section: Methodsmentioning

confidence: 99%

Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain

Noeske

Schwalbe

Wöhnert

2007

127

120

Divalent cations are important in the folding and stabilization of complex RNA structures. The adenine-sensing riboswitch controls the expression of mRNAs for proteins involved in purine metabolism by directly sensing intracellular adenine levels. Adenine binds with high affinity and specificity to the ligand binding or aptamer domain of the adenine-sensing riboswitch. The X-ray structure of this domain in complex with adenine revealed an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base-pairing interactions and identified five binding sites for hexahydrated Mg2+-ions. Furthermore, a role for Mg2+-ions in the ligand-induced folding of this RNA was suggested. Here, we describe the interaction of divalent cations with the RNA–adenine complex in solution as studied by high-resolution NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping and intermolecular nuclear Overhauser effects (NOEs) indicate the presence of at least three binding sites for divalent cations. Two of them are similar to those in the X-ray structure. The third site, which is important for the folding of this RNA, has not been observed previously. The ligand-free state of the RNA is conformationally heterogeneous and contains base-pairing patterns detrimental to ligand binding in the absence of Mg2+, but becomes partially pre-organized for ligand binding in the presence of Mg2+. Compared to the highly similar guanine-sensing riboswitch, the folding pathway for the adenine-sensing riboswitch aptamer domain is more complex and the influence of Mg2+ is more pronounced.

“…Chemical shifts were referenced as described previously (21). Resonance assignment of the exchangeable and non-exchangeable protons of BEVSLDap was performed using ( 1 H, 15 N)-HSQC (22), ( 1 H, 15 N)-CPMG-NOESY (23), ( 1 H, 1 H)-H5(C5C4N)H (C,U) (24), ( 1 H, 1 H)-HCCNH-TOCSY (G) (25), ( 1 H, 1 H)-HCCH-TOCSY (A) (26), ( 1 H, 1 H)-HCCH-COSY (C,U) (27), ( 1 H, 15 N)-2bond-HSQC (28), ( 1 H, 13 C)-H(N)CO (29), ( 1 H, 13 C)-HSQC, 3D ( 1 H, 1 H, 13 C)-HCCH-COSY (sugar), 3D ( 1 H, 1 H, 13 C)-HCCH-TOCSY (sugar) (27) and ( 1 H, 15 N)-HCN (30). For sequential assignments 3D ( 1 H, 1 H, 13 C)-NOESY-HSQC (31) and ( 1 H, 31 P)-HCP-CCH-TOCSY (32) spectra were acquired.…”

Section: Methodsmentioning

confidence: 99%

A novel cGUUAg tetraloop structure with a conserved yYNMGg-type backbone conformation from cloverleaf 1 of bovine enterovirus 1 RNA

Ihle

Ohlenschläger²,

Häfner³

et al. 2005

The 5′-terminal cloverleaf (CL)-like RNA structures are essential for the initiation of positive- and negative-strand RNA synthesis of entero- and rhinoviruses. SLD is the cognate RNA ligand of the viral proteinase 3C (3Cpro), which is an indispensable component of the viral replication initiation complex. The structure of an 18mer RNA representing the apical stem and the cGUUAg D-loop of SLD from the first 5′-CL of BEV1 was determined in solution to a root-mean-square deviation (r.m.s.d.) (all heavy atoms) of 0.59 Å (PDB 1Z30). The first (antiG) and last (synA) nucleotide of the D-loop forms a novel ‘pseudo base pair’ without direct hydrogen bonds. The backbone conformation and the base-stacking pattern of the cGUUAg-loop, however, are highly similar to that of the coxsackieviral uCACGg D-loop (PDB 1RFR) and of the stable cUUCGg tetraloop (PDB 1F7Y) but surprisingly dissimilar to the structure of a cGUAAg stable tetraloop (PDB 1MSY), even though the cGUUAg BEV D-loop and the cGUAAg tetraloop differ by 1 nt only. Together with the presented binding data, these findings provide independent experimental evidence for our model [O. Ohlenschläger, J. Wöhnert, E. Bucci, S. Seitz, S. Häfner, R. Ramachandran, R. Zell and M. Görlach (2004) Structure, 12, 237–248] that the proteinase 3Cpro recognizes structure rather than sequence.