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- Zentrum für Biomolekulare Magnetische Resonanz (BMRZ) (4) (remove)
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.
Herein, we present a multi-cycle chemoenzymatic synthesis of modified RNA with simplified solid-phase handling to overcome size limitations of RNA synthesis. It combines the advantages of classical chemical solid-phase synthesis and enzymatic synthesis using magnetic streptavidin beads and biotinylated RNA. Successful introduction of light-controllable RNA nucleotides into the tRNAMet sequence was confirmed by gel electrophoresis and mass spectrometry. The methods tolerate modifications in the RNA phosphodiester backbone and allow introductions of photocaged and photoswitchable nucleotides as well as photocleavable strand breaks and fluorophores.
PELDOR (pulse electron-electron double resonance) is an established method to study intramolecular distances and can give evidence for conformational changes and flexibilities. However, it can also be used to study intermolecular interactions as for example oligerimization. Here, we used PELDOR to study the ‘end-to-end’ stacking of small double stranded (ds)RNAs. For this study, the dsRNA molecules were only singly labelled with the spin label TPA to avoid multi-spin effects and to measure only the intermolecular stacking interactions. It can be shown that small dsRNAs tend to assemble to rod-like structures due to π-π-interactions between the base pairs at the end of the strands. On the one hand, these interactions can influence or complicate measurements aimed at the determining of the structure and dynamics of the dsRNA molecule itself. On the other hand, it can be interesting to study such intermolecular stacking interactions in more detail, as for example their dependence on ion concentration. We quantitatively determined the stacking probability as a function of the monovalent NaCl salt and the dsRNA concentration. From this data the dissociation constant Kd was deduced and found to depend on the ratio between the NaCl salt and dsRNA concentrations. Additionally, the distances and distance distributions obtained predict a model for the stacking geometry of dsRNAs. Introducing a nucleotide overhangs at one end of the dsRNA molecule restricts the stacking to the other end, leading only to dimer formations. Introducing such an overhang at both ends of the dsRNA molecule fully suppresses stacking, as we could demonstrate by PELDOR experiments quantitatively.
Global response of diacylglycerol kinase towards substrate binding observed by 2D and 3D MAS NMR
(2019)
Escherichia coli diacylglycerol kinase (DGK) is an integral membrane protein, which catalyses the ATP-dependent phosphorylation of diacylglycerol (DAG) to phosphatic acid (PA). It is a unique trimeric enzyme, which does not share sequence homology with typical kinases. It exhibits a notable complexity in structure and function despite of its small size. Here, chemical shift assignment of wild-type DGK within lipid bilayers was carried out based on 3D MAS NMR, utilizing manual and automatic analysis protocols. Upon nucleotide binding, extensive chemical shift perturbations could be observed. These data provide evidence for a symmetric DGK trimer with all of its three active sites concurrently occupied. Additionally, we could detect that the nucleotide substrate induces a substantial conformational change, most likely directing DGK into its catalytic active form. Furthermore, functionally relevant interprotomer interactions are identified by DNP-enhanced MAS NMR in combination with site-directed mutagenesis and functional assays.