Reconstitution of Pyrobaculum aerophilum RNase P, a catalytic ribonucleoprotein involved in tRNA maturation
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Date
2011-06
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The Ohio State University
Abstract
RNase P, an essential ribonucleoprotein (RNP) complex, is responsible for the processing and removal of 5′ tRNA leaders. While RNase P-catalyzed tRNA maturation is common to the three domains of life, the subunit make-up of this enzyme is quite different. The RNase P holoenzyme is composed of one RNase P RNA (RPR) and a variable number of RNase P Protein (RPP) subunits depending on the source: one in bacteria, up to five in archaea, and up to ten in eukarya. While the structurally related RPR is the catalytic moiety in all these RNPs, none of the RPPs (all of which are essential in vivo) can catalyze precursor tRNA processing independently of the RPR. Thus, RNase P is a paradigm for the intimate cooperation of RNA and protein subunits necessary to generate an efficient biological catalyst. The failure to reconstitute eukaryotic RNase P (one RPR + ≤ 10 RPPs) has led to the use of archaeal RNase P (one RPR + ≤ 5 RPPs, which share eukaryotic homologs) as an experimental surrogate for studying the functional interplay among multiple protein subunits and a single RNA catalyst. Recent work has demonstrated that Pyrobaculum aerophilum (Pae), an ancient hyper-thermophilic crenarchaeon, might yet have the smallest natural RPR (207 nucleotides) and only four of the five expected archaeal RPPs.
In this work, the genes of four Pae RPPs (POP5, RPP29, RPP30, and L7Ae) were cloned from Pae genomic DNA. The first 20 codons of POP5, RPP29, and RPP30 were optimized for protein overexpression in Escherichia coli (Eco) since the native Pae archaeal sequences did not support robust expression in bacteria. POP5 and RPP30 were co-overexpressed as a binary complex in Eco SHuffle cells. RPP29 and L7Ae were overexpressed in Eco SHuffle and Eco BL21(DE3) Rosetta cells, respectively. Successful purification schemes were developed for all four Pae RPPs. Ultimately, functional reconstitution of the Pae RNase P holoenzyme using in vitro transcribed RPR and purified RPPs was achieved.
Overall, Pae RNase P is a distinctive variant among its counterparts and this study provides a foundation for dissecting its structure-function relationships and gaining insights into the possible transfer of some functions from the RNA to protein subunits during the evolution of RNase P.
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RNase P, tRNA maturation, Pyrobaculum aerophilum, ribonuclease P