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Nep1 (Emg1) is a highly conserved nucleolar protein with an essential function in ribosome biogenesis. A mutation in the human Nep1 homolog causes Bowen–Conradi syndrome—a severe developmental disorder. Structures of Nep1 revealed a dimer with a fold similar to the SPOUT-class of RNA-methyltransferases suggesting that Nep1 acts as a methyltransferase in ribosome biogenesis. The target for this putative methyltransferase activity has not been identified yet. We characterized the RNA-binding specificity of Methanocaldococcus jannaschii Nep1 by fluorescence- and NMR-spectroscopy as well as by yeast three-hybrid screening. Nep1 binds with high affinity to short RNA oligonucleotides corresponding to nt 910–921 of M. jannaschii 16S rRNA through a highly conserved basic surface cleft along the dimer interface. Nep1 only methylates RNAs containing a pseudouridine at a position corresponding to a previously identified hypermodified N1-methyl-N3-(3-amino-3-carboxypropyl) pseudouridine (m1acp3-Psi) in eukaryotic 18S rRNAs. Analysis of the methylated nucleoside by MALDI-mass spectrometry, HPLC and NMR shows that the methyl group is transferred to the N1 of the pseudouridine. Thus, Nep1 is the first identified example of an N1-specific pseudouridine methyltransferase. This enzymatic activity is also conserved in human Nep1 suggesting that Nep1 is the methyltransferase in the biosynthesis of m1acp3-Psi in eukaryotic 18S rRNAs.
The mfl-riboswitch regulates expression of ribonucleotide reductase subunit in Mesoplasma florum by binding to 2´-deoxyguanosine and thereby promoting transcription termination. We characterized the structure of the ligand-bound aptamer domain by NMR spectroscopy and compared the mfl-aptamer to the aptamer domain of the closely related purine-sensing riboswitches. We show that the mfl-aptamer accommodates the extra 2´-deoxyribose unit of the ligand by forming a more relaxed binding pocket than these found in the purine-sensing riboswitches. Tertiary structures of the xpt-aptamer bound to guanine and of the mfl-aptamer bound to 2´-deoxyguanosine exhibit very similar features, although the sequence of the mfl-aptamer contains several alterations compared to the purine-aptamer consensus sequence. These alterations include the truncation of a hairpin loop which is crucial for complex formation in all purine-sensing riboswitches characterized to date. We further defined structural features and ligand binding requirements of the free mfl-aptamer and found that the presence of Mg2+ is not essential for complex formation, but facilitates ligand binding by promoting pre-organization of key structural motifs in the free aptamer.
In bacteria, the regulation of gene expression by cis-acting transcriptional riboswitches located in the 5'-untranslated regions of messenger RNA requires the temporal synchronization of RNA synthesis and ligand binding-dependent conformational refolding. Ligand binding to the aptamer domain of the riboswitch induces premature termination of the mRNA synthesis of ligand-associated genes due to the coupled formation of 3'-structural elements acting as terminators. To date, there has been no high resolution structural description of the concerted process of synthesis and ligand-induced restructuring of the regulatory RNA element. Here, we show that for the guanine-sensing xpt-pbuX riboswitch from Bacillus subtilis, the conformation of the full-length transcripts is static: it exclusively populates the functional off-state but cannot switch to the on-state, regardless of the presence or absence of ligand. We show that only the combined matching of transcription rates and ligand binding enables transcription intermediates to undergo ligand-dependent conformational refolding.
In a combined NMR/MD study, the temperature-dependent changes in the conformation of two members of the RNA YNMG-tetraloop motif (cUUCGg and uCACGg) have been investigated at temperatures of 298, 317 and 325 K. The two members have considerable different thermal stability and biological functions. In order to address these differences, the combined NMR/MD study was performed. The large temperature range represents a challenge for both, NMR relaxation analysis (consistent choice of effective bond length and CSA parameter) and all-atom MD simulation with explicit solvent (necessity to rescale the temperature). A convincing agreement of experiment and theory is found. Employing a principle component analysis of the MD trajectories, the conformational distribution of both hairpins at various temperatures is investigated. The ground state conformation and dynamics of the two tetraloops are indeed found to be very similar. Furthermore, both systems are initially destabilized by a loss of the stacking interactions between the first and the third nucleobase in the loop region. While the global fold is still preserved, this initiation of unfolding is already observed at 317 K for the uCACGg hairpin but at a significantly higher temperature for the cUUCGg hairpin.
Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain
(2007)
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.