Functional reconstitution and characterization of Pyrococcus furiosus RNase P
Journal
Proceedings of the National Academy of Sciences of the United States of America
Journal Volume
103
Journal Issue
44
Pages
16147
Date Issued
2006
Author(s)
Abstract
RNase P, which catalyzes the magnesium-dependent 5′-end maturation of tRNAs in all three domains of life, is composed of one essential RNA and a varying number of protein subunits depending on the source: at least one in bacteria, four in archaea, and nine in eukarya. To address why multiple protein subunits are needed for archaeal eukaryal RNase P catalysis, in contrast to their bacterial relative, in vitro reconstitution of these holoenzymes is a prerequisite. Using recombinant subunits, we have reconstituted in vitro the RNase P holoenzyme from the thermophilic archaeon Pyroccocus furiosus (Pfu) and furthered our understanding regarding its functional organization and assembly pathway(s). Whereas Pfu RNase P RNA (RPR) alone is capable of multiple turnover, addition of all four RNase P protein (Rpp) subunits to Pfu RPR results in a 25-fold increase in its kcat and a 170-fold decrease in K m. In fact, even in the presence of only one of two specific pairs of Rpps, the RPR displays activity at lower substrate and magnesium concentrations. Moreover, a pared-down, mini-Pfu RNase P was identified with an RPR deletion mutant. Results from our kinetic and foot-printing studies on Pfu RNase P, together with insights from recent structures of bacterial RPRs, provide a framework for appreciating the role of multiple Rpps in archaeal RNase P. © 2006 by The National Academy of Sciences of the USA.
Subjects
archaeal RNase P; in vitro reconstitution; precursor tRNA processing
SDGs
Publisher
NATL ACAD SCIENCES
Type
journal article