Functional reconstitution and characterization of Pyrococcus furiosus RNase P. Proc Natl Acad Sci U S A 2006 Oct 31;103(44):16147-52
Date
10/21/2006Pubmed ID
17053064Pubmed Central ID
PMC1637551DOI
10.1073/pnas.0608000103Scopus ID
2-s2.0-33750831489 (requires institutional sign-in at Scopus site) 82 CitationsAbstract
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 Pyrococcus 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 k(cat) 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 footprinting 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.
Author List
Tsai HY, Pulukkunat DK, Woznick WK, Gopalan VAuthor
Walter Woznick MD Assistant Professor in the Medicine department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Base SequenceBinding Sites
Catalysis
Escherichia coli
Holoenzymes
Kinetics
Molecular Sequence Data
Nucleic Acid Conformation
Protein Binding
Protein Subunits
Pyrococcus furiosus
RNA, Transfer
Recombinant Proteins
Ribonuclease P
