Abstract
Ribonuclease P (RNase P) is responsible for the generation of mature 5' termini of tRNA. The RNA component of this complex encodes the enzymatic activity in bacteria and is itself catalytically active under appropriate conditions in vitro. The role of the subunits in eucaryotes has not yet been established. We have partially purified RNase P activity from the ciliate protozoan Tetrahymena thermophila to learn more about the biochemical characteristics of RNase P from a lower eucaryote. The Tetrahymena RNase P displays a pH optimum and temperature optimum characteristic of RNase P enzymes isolated from other organisms. The Km of the T. thermophila enzyme for pre-tRNAGln is 1.6 x 10(-7)M, which is comparable to the values reported for other examples of RNase P. The Tetrahymena RNase P is a ribonucleoprotein complex, as supported by its sensitivity to micrococcal nuclease and proteinase K. The buoyant density of the enzyme in Cs2SO4 is 1.42 g/ml, which suggests that the RNA component of the Tetrahymena enzyme comprises a significantly greater percentage of the holoenzyme than that determined for RNase P of other Eucarya or Archaea. The holoenzyme has a requirement for divalent cations displaying characteristics that are unique for RNase P but closely resemble preferences reported for the Tetrahymena group I intron RNA. Puromycin inhibits pre-tRNA processing by the Tetrahymena complex, and implications of the similarities between recognition of tRNA by ribosomal components and RNase P are discussed.
Highlights
This study describes the isolation and characterization of Ribonuclease P (RNase P) from a ciliate protozoan
RNase P from other eucaryotic organisms has been purified by chromatography on successive anion and cation exchange resins [11, 13, 14, 16, 17]
The application of hydrophobic resins, glycerol gradient sedimentation, and Cs2SO4 density gradient centrifugation, each of which had been successfully used during the purification of other eucaryotic RNase P enzymes [9, 31], failed to afford us either substantial or stable enrichment of the T. thermophila enzyme
Summary
(Received for publication, January 18, 1996, and in revised form, April 3, 1996). Heather L. Phylogenetic analyses of the components of the bacterial RNase P holoenzyme suggest that both the RNA and protein subunit genes change more rapidly than the corresponding ribosomal RNA gene sequences [1, 20]. We have chosen to focus our studies on the RNase P istics that are unique for RNase P but closely resemble holoenzymes from ciliate protozoa because these organisms preferences reported for the Tetrahymena group I in- comprise an extremely phylogenetically diverse group of orgatron RNA. Through the examination of the biochemical and molecular properties of homologous enzymes from phylogenetically diverse origins, considerable insight can be obtained relevant to the catalytic mechanism of RNase P and the role of the molecular components in holoenzyme function. The endoribonuclease ribonuclease P (RNase P) cleaves the nascent transcript
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