Functional reconstitution and characterization of Pyrococcus furiosus RNase P.
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.
Tsai
Hsin-Yue
HY
Molecular, Cellular and Developmental Biology Graduate Program, Ohio State Biochemistry Program, Ohio State University, Columbus, OH 43210, USA.
Pulukkunat
Dileep K
DK
Woznick
Walter K
WK
Gopalan
Venkat
V
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D043262:Ribonuclease P / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D020035:Holoenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; D012343:RNA, Transfer; D043262:Ribonuclease P