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  • 标题:NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P
  • 本地全文:下载
  • 作者:Kristin S. Koutmou ; Anette Casiano-Negroni ; Melissa M. Getz
  • 期刊名称:Proceedings of the National Academy of Sciences
  • 印刷版ISSN:0027-8424
  • 电子版ISSN:1091-6490
  • 出版年度:2010
  • 卷号:107
  • 期号:6
  • 页码:2479-2484
  • DOI:10.1073/pnas.0906319107
  • 语种:English
  • 出版社:The National Academy of Sciences of the United States of America
  • 摘要:Functionally critical metals interact with RNA through complex coordination schemes that are currently difficult to visualize at the atomic level under solution conditions. Here, we report a new approach that combines NMR and XAS to resolve and characterize metal binding in the most highly conserved P4 helix of ribonuclease P (RNase P), the ribonucleoprotein that catalyzes the divalent metal ion-dependent maturation of the 5' end of precursor tRNA. Extended X-ray absorption fine structure (EXAFS) spectroscopy reveals that the Zn2+ bound to a P4 helix mimic is six-coordinate, with an average Zn-O/N bond distance of 2.08 A. The EXAFS data also show intense outer-shell scattering indicating that the zinc ion has inner-shell interactions with one or more RNA ligands. NMR Mn2+ paramagnetic line broadening experiments reveal strong metal localization at residues corresponding to G378 and G379 in B. subtilis RNase P. A new "metal cocktail" chemical shift perturbation strategy involving titrations with [IMG]/medium/pnas.0906319107eq1.gif" ALT="Formula "> , Zn2+, and [IMG]/medium/pnas.0906319107eq2.gif" ALT="Formula "> confirm an inner-sphere metal interaction with residues G378 and G379. These studies present a unique picture of how metals coordinate to the putative RNase P active site in solution, and shed light on the environment of an essential metal ion in RNase P. Our experimental approach presents a general method for identifying and characterizing inner-sphere metal ion binding sites in RNA in solution.
  • 关键词:manganese ; ribozyme ; RNase P ; X-ray absorption spectroscopy ; zinc
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