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1.
J Am Chem Soc ; 132(5): 1454-5, 2010 Feb 10.
Article in English | MEDLINE | ID: mdl-20078041

ABSTRACT

Pulsed electron double resonance (PELDOR) spectroscopy reveals a prearranged tertiary structure of the 27 nucleotides long engineered neomycin-responsive riboswitch. Measured distances between spin labels at positions U4-U14, U4-U15, U14-U26, and U15-U26 were unchanged upon neomycin binding which implies that the global stem-loop architecture is preserved in the absence and presence of the ligand. On the basis of our results, we infer that low-temperature PELDOR data unambiguously demonstrate the existence of an enthalpically favorable set of RNA conformations ready to bind the ligand without major global rearrangement.


Subject(s)
Anti-Bacterial Agents/metabolism , Electron Spin Resonance Spectroscopy/methods , Neomycin/metabolism , RNA/analysis , Nucleic Acid Conformation , Protein Binding , RNA/metabolism , Spin Labels , Uridine/analysis
2.
Nucleic Acids Res ; 38(7): 2387-98, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20047967

ABSTRACT

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.


Subject(s)
Archaeal Proteins/chemistry , Methanococcales/enzymology , Methyltransferases/chemistry , Nuclear Proteins/chemistry , Pseudouridine/metabolism , RNA, Ribosomal/metabolism , Archaeal Proteins/metabolism , Base Sequence , Binding Sites , Consensus Sequence , Humans , Methanococcales/genetics , Methylation , Methyltransferases/metabolism , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/metabolism , Pseudouridine/analogs & derivatives , Pseudouridine/analysis , RNA, Fungal/chemistry , RNA, Fungal/metabolism , RNA, Ribosomal/chemistry , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Spectrometry, Fluorescence , Two-Hybrid System Techniques
3.
Acta Crystallogr Sect E Struct Rep Online ; 65(Pt 3): o529, 2009 Feb 13.
Article in English | MEDLINE | ID: mdl-21582190

ABSTRACT

The six-membered ring of the title compound, C(11)H(16)NO, has a distorted envelope conformation. The piperidine N atom deviates by 0.128 (1) Šfrom the plane through its three neighbouring atoms. In the crystal structure, mol-ecules are connected by inter-molecular C(ethyn-yl)-H⋯O contacts to form chains extending in the [10] direction.

4.
Acta Crystallogr Sect E Struct Rep Online ; 65(Pt 8): o1848, 2009 Jul 11.
Article in English | MEDLINE | ID: mdl-21583548

ABSTRACT

The five-membered ring of the title compound, C(10)H(14)NO, is almost planar [mean deviation from best plane = 0.006 (1) Å]. The N-O bond is in the plane of the five-membered ring. The mol-ecule is positioned about a pseudo-mirror plane at y = 0.375. In the crystal, mol-ecules are connected by inter-molecular C-H⋯O contacts into layers parallel to (010).

5.
Article in English | MEDLINE | ID: mdl-18066874

ABSTRACT

For EPR measurements of RNA, DNA, or proteins, the occurrence of the paramagnetic species is necessary. The aim of this work is to improve the synthesis of two different EPR spinlabels 2,2,6,6-tetra methyl-3,4-dehydro-piperidin-N-oxyl-4-acetylene (TEMPA) 6 and 15N-labeled TEMPA 6* and their coupling to uridine. The yield of the synthesis of TEMPA could be increased to 40% and the second nitroxide 2,2,6,6-tetramethyl-3,4-dehydro-piperidin-15N-oxyl-4-acetylene 6* could be synthesized with a yield of 11%.


Subject(s)
Electron Spin Resonance Spectroscopy , Spin Labels/chemical synthesis , Uridine/chemistry , Chromatography, High Pressure Liquid
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