NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P.

Published

Journal Article

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 Zn(2+) 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 Mn(2+) 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 , Zn(2+), and 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.

Full Text

Duke Authors

Cited Authors

  • Koutmou, KS; Casiano-Negroni, A; Getz, MM; Pazicni, S; Andrews, AJ; Penner-Hahn, JE; Al-Hashimi, HM; Fierke, CA

Published Date

  • February 9, 2010

Published In

Volume / Issue

  • 107 / 6

Start / End Page

  • 2479 - 2484

PubMed ID

  • 20133747

Pubmed Central ID

  • 20133747

Electronic International Standard Serial Number (EISSN)

  • 1091-6490

Digital Object Identifier (DOI)

  • 10.1073/pnas.0906319107

Language

  • eng

Conference Location

  • United States