%0 Journal Article
%T Assembly of the Complex between Archaeal RNase P Proteins RPP30 and Pop5
%A Brandon L. Crowe
%A Christopher J. Bohlen
%A Ross C. Wilson
%A Venkat Gopalan
%A Mark P. Foster
%J Archaea
%D 2011
%I Hindawi Publishing Corporation
%R 10.1155/2011/891531
%X 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每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 (25ˋkDa) 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 (14ˋkDa) reveal its binding surface on RPP30. ITC experiments confirm a net 1ˋ:ˋ1 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 78ˋkDa 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. 1. Introduction Ribonuclease P (RNase P) is a ribonucleoprotein (RNP) complex primarily responsible for cleaving the 5∩ leader sequence of precursor-tRNA (pre-tRNA) molecules in all domains of life [1每3]. The RNase P RNA subunit (RPR) constitutes the Mg2+-dependent catalytic moiety and supports pre-tRNA cleavage on its own in vitro [4每6]. The bacterial RNase P holoenzyme contains one large RPR and one conserved protein (RNase P protein, RPP) that is essential for function in vivo [7]. The bacterial RPP aids RPR catalysis by increasing the affinity of the holoenzyme for the substrate and for the Mg2+ cofactor [8每11]. Eukaryal and archaeal genomes do not encode sequence homologs of the bacterial RPP [12]. Instead, eukaryal and archaeal RNase P holoenzymes comprise multiple RPPs (up to 10 in eukarya and 5 in archaea) together with an RPR [12, 13]. The known archaeal RPPs are homologous to eukaryal proteins Pop5, RPP21, RPP29, RPP30, and RPP38 [21每23]. Recombinantly expressed RPPs have been assembled with the in vitro transcribed cognate RPR to reconstitute the holoenzyme from several archaea, including Methanothermobacter thermoautotrophicus (Mth) [24, 25] Pyrococcus horikoshii (Pho) [23], Pyrococcus furiosus (Pfu) [26], Methanocaldococcus jannaschii (Mja) [25, 27], and
%U http://www.hindawi.com/journals/archaea/2011/891531/