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Archaea
Volume 2011 (2011), Article ID 891531, 12 pages
http://dx.doi.org/10.1155/2011/891531
Research Article

Assembly of the Complex between Archaeal RNase P Proteins RPP30 and Pop5

1Department of Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH 43210-1214, USA
2Ohio State Biochemistry Program, Ohio State University, Columbus, OH 43210-1214, USA

Received 2 March 2011; Revised 10 August 2011; Accepted 17 August 2011

Academic Editor: Herman van Tilbeurgh

Copyright © 2011 Brandon L. Crowe et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

RNase P is a highly conserved ribonucleoprotein enzyme that represents a model complex for understanding macromolecular RNA-protein interactions. Archaeal RNase P consists of one RNA and up to five proteins (Pop5, RPP30, RPP21, RPP29, and RPP38/L7Ae). Four of these proteins function in pairs (Pop5-RPP30 and RPP21–RPP29). We have used nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to characterize the interaction between Pop5 and RPP30 from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu). NMR backbone resonance assignments of free RPP30 (25 kDa) indicate that the protein is well structured in solution, with a secondary structure matching that observed in a closely related crystal structure. Chemical shift perturbations upon the addition of Pop5 (14 kDa) reveal its binding surface on RPP30. ITC experiments confirm a net 1 : 1 stoichiometry for this tight protein-protein interaction and exhibit complex isotherms, indicative of higher-order binding. Indeed, light scattering and size exclusion chromatography data reveal the complex to exist as a 78 kDa heterotetramer with two copies each of Pop5 and RPP30. These results will inform future efforts to elucidate the functional role of the Pop5-RPP30 complex in RNase P assembly and catalysis.