Nejvíce citovaný článek - PubMed ID 12061713
Determination of the glycosidic torsion angles in uniformly 13C-labeled nucleic acids from vicinal coupling constants 3J(C2)/4-H1' and 3J(C6)/8-H1'
Heteronuclear and homonuclear direct (D) and indirect (J) spin-spin interactions are important sources of structural information about nucleic acids (NAs). The Hamiltonians for the D and J interactions have the same functional form; thus, the experimentally measured apparent spin-spin coupling constant corresponds to a sum of J and D. In biomolecular NMR studies, it is commonly presumed that the dipolar contributions to Js are effectively canceled due to random molecular tumbling. However, in strong magnetic fields, such as those employed for NMR analysis, the tumbling of NA fragments is anisotropic because the inherent magnetic susceptibility of NAs causes an interaction with the external magnetic field. This motional anisotropy is responsible for non-zero D contributions to Js. Here, we calculated the field-induced D contributions to 33 structurally relevant scalar coupling constants as a function of magnetic field strength, temperature and NA fragment size. We identified two classes of Js, namely (1)JCH and (3)JHH couplings, whose quantitative interpretation is notably biased by NA motional anisotropy. For these couplings, the magnetic field-induced dipolar contributions were found to exceed the typical experimental error in J-coupling determinations by a factor of two or more and to produce considerable over- or under-estimations of the J coupling-related torsion angles, especially at magnetic field strengths >12 T and for NA fragments longer than 12 bp. We show that if the non-zero D contributions to J are not properly accounted for, they might cause structural artifacts/bias in NA studies that use solution NMR spectroscopy.
- Klíčová slova
- Dipolar coupling, Karplus equation, Magnetic susceptibility, Nucleic acid, Scalar coupling, Self-alignment,
- MeSH
- kvantová teorie MeSH
- magnetické pole * MeSH
- nukleární magnetická rezonance biomolekulární metody MeSH
- nukleové kyseliny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- nukleové kyseliny MeSH
Non-coding RNA polymerase II transcripts are processed by the poly(A)-independent termination pathway that requires the Nrd1 complex. The Nrd1 complex includes two RNA-binding proteins, the nuclear polyadenylated RNA-binding (Nab) 3 and the nuclear pre-mRNA down-regulation (Nrd) 1 that bind their specific termination elements. Here we report the solution structure of the RNA-recognition motif (RRM) of Nab3 in complex with a UCUU oligonucleotide, representing the Nab3 termination element. The structure shows that the first three nucleotides of UCUU are accommodated on the β-sheet surface of Nab3 RRM, but reveals a sequence-specific recognition only for the central cytidine and uridine. The specific contacts we identified are important for binding affinity in vitro as well as for yeast viability. Furthermore, we show that both RNA-binding motifs of Nab3 and Nrd1 alone bind their termination elements with a weak affinity. Interestingly, when Nab3 and Nrd1 form a heterodimer, the affinity to RNA is significantly increased due to the cooperative binding. These findings are in accordance with the model of their function in the poly(A) independent termination, in which binding to the combined and/or repetitive termination elements elicits efficient termination.
- MeSH
- genetická transkripce * MeSH
- jaderné proteiny chemie genetika metabolismus MeSH
- konformace proteinů MeSH
- magnetická rezonanční spektroskopie MeSH
- multimerizace proteinu MeSH
- oligonukleotidy chemie metabolismus MeSH
- proteiny vázající RNA chemie genetika metabolismus MeSH
- roztoky MeSH
- Saccharomyces cerevisiae - proteiny chemie genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika MeSH
- sekvence nukleotidů MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- jaderné proteiny MeSH
- NAB3 protein, S cerevisiae MeSH Prohlížeč
- NRD1 protein, S cerevisiae MeSH Prohlížeč
- oligonukleotidy MeSH
- proteiny vázající RNA MeSH
- roztoky MeSH
- Saccharomyces cerevisiae - proteiny MeSH
We describe a novel, fundamental property of nucleobase structure, namely, pyramidilization at the N1/9 sites of purine and pyrimidine bases. Through a combined analyses of ultra-high-resolution X-ray structures of both oligonucleotides extracted from the Nucleic Acid Database and isolated nucleotides and nucleosides from the Cambridge Structural Database, together with a series of quantum chemical calculations, molecular dynamics (MD) simulations, and published solution nuclear magnetic resonance (NMR) data, we show that pyramidilization at the glycosidic nitrogen is an intrinsic property. This property is common to isolated nucleosides and nucleotides as well as oligonucleotides-it is also common to both RNA and DNA. Our analysis suggests that pyramidilization at N1/9 sites depends in a systematic way on the local structure of the nucleoside. Of note, the pyramidilization undergoes stereo-inversion upon reorientation of the glycosidic bond. The extent of the pyramidilization is further modulated by the conformation of the sugar ring. The observed pyramidilization is more pronounced for purine bases, while for pyrimidines it is negligible. We discuss how the assumption of nucleic acid base planarity can lead to systematic errors in determining the conformation of nucleotides from experimental data and from unconstrained MD simulations.
- MeSH
- deoxyadenosiny chemie MeSH
- deoxycytidin chemie MeSH
- dusík chemie MeSH
- krystalografie rentgenová MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- oligonukleotidy chemie MeSH
- počítačová simulace MeSH
- purinové nukleosidy chemie MeSH
- purinové nukleotidy chemie MeSH
- puriny chemie MeSH
- pyrimidinové nukleosidy chemie MeSH
- pyrimidinové nukleotidy chemie MeSH
- pyrimidiny chemie MeSH
- sacharidy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 2'-deoxyadenosine MeSH Prohlížeč
- deoxyadenosiny MeSH
- deoxycytidin MeSH
- dusík MeSH
- oligonukleotidy MeSH
- purinové nukleosidy MeSH
- purinové nukleotidy MeSH
- puriny MeSH
- pyrimidinové nukleosidy MeSH
- pyrimidinové nukleotidy MeSH
- pyrimidiny MeSH
- sacharidy MeSH
DNA A-tracts have been defined as four or more consecutive A.T base pairs without a TpA step. When inserted in phase with the DNA helical repeat, bending is manifested macroscopically as anomalous migration on polyacrylamide gels, first observed >20 years ago. An unsolved conundrum is why DNA containing in-phase A-tract repeats of A(4)T(4) are bent, whereas T(4)A(4) is straight. We have determined the solution structures of the DNA duplexes formed by d(GCAAAATTTTGC) [A4T4] and d(CGTTTTAAAACG) [T4A4] with NH(4)(+) counterions by using NMR spectroscopy, including refinement with residual dipolar couplings. Analysis of the structures shows that the ApT step has a large negative roll, resulting in a local bend toward the minor groove, whereas the TpA step has a positive roll and locally bends toward the major groove. For A4T4, this bend is nearly in phase with bends at the two A-tract junctions, resulting in an overall bend toward the minor groove of the A-tract, whereas for T4A4, the bends oppose each other, resulting in a relatively straight helix. NMR-based structural modeling of d(CAAAATTTTG)(15) and d(GTTTTAAAAC)(15) reveals that the former forms a left-handed superhelix with a diameter of approximately 110 A and pitch of 80 A, similar to DNA in the nucleosome, whereas the latter has a gentle writhe with a pitch of >250 A and diameter of approximately 50 A. Results of gel electrophoretic mobility studies are consistent with the higher-order structure of the DNA and furthermore depend on the nature of the monovalent cation present in the running buffer.
