Archaeal Ribonuclease P Has Multiple Protein Subunits Homologous to Eukaryotic Nuclear Ribonuclease P Subunits

HALL, THOMAS A. Archaeal Ribonuclease P has Multiple Protein Subunits Homologous to Eukaryotic Nuclear Ribonuclease P Subunits (Under the direction of James W. Brown.) RNase P removes the 5 ́ leader from all transfer RNA precursors, and is thus a required factor for protein synthesis. RNase P consists of a catalytic RNA molecule and associated protein, the amount of which varies among phylogenetic domains. The ca. 120 kDa RNA component of RNase P from Bacteria is catalytic in vitro in the absence of its single, small (14 kDa) protein cofactor. RNase P from the eukaryotic nucleus, which has more protein (nine proteins in yeast) has an RNA that displays no catalytic activity without its protein. The Archaea, which resemble the Eukarya more than the Bacteria in their transcription and translation apparatus, have RNase P RNAs almost identical in secondary structure to those of Bacteria. However, many of these RNAs display no catalytic activity in vitro without their proteins, and those that are catalytic require extraordinarily high concentrations of both monovalent and divalent salts. Previous characterizations of archaeal RNase P have presented conflicting and somewhat confusing results. RNase P from Sulfolobus acidocaldarius (Crenarchaea) has a buoyant density in Cs2SO4 of 1.27 g/ml, an apparent exclusion size of 400 kDa, and is resistant to micrococcal nuclease. RNase P from Haloferax volcanii (Euryarchaea), has a density of 1.61 g/ml in Cs2SO4, an apparent exclusion size of 165 kDa, and is sensitive to micrococcal nuclease. Despite these differences, both of these enzymes have RNAs with structures similar to the bacterial counterpart. Methanothermobacter thermoautotrophicus (formerly Methanobacterium thermoautotrophicum strain ∆H) is a moderately deeply branching methanogen roughly intermediate in phylogenetic trees between extremes halophiles (e.g. H. volcanii) and the Crenarchaea (e.g. S. acidocaldarius). It has an RNA that is strikingly similar to bacterial RNase P RNAs in structure, but requires about 3M monovalent cations and 300 mM Mg for catalytic activity in vitro without its protein cofactor, and the Km of this reaction is more than 1000-fold higher than for the holoenzyme. The protein subunit of RNase P from Bacillus subtilis can reconstitute with the RNase RNA from Methanobacteria at low ionic strengths. Despite this observation, there is no open reading frame in the M. thermoautotrophicus genome with similarity to a bacterial RNase P subunit, and its RNase P has a buoyant density in Cs2SO4 of 1.42 g/ml, suggesting more protein than bacterial RNase P. There are, however, four open reading frames (ORFs) in the genome of M. thermoautotrophicus, MTH11, MTH687, MTH688 and MTH1618, with recognizable similarity to the four recently identified yeast RNase P subunits Pop4p, Pop5p, Rpp1p and Rpr2p, respectively, and these ORFs have homologs in other Archaea. Antibodies generated to the recombinant versions of these proteins detect all four proteins in western blots of purified M. thermoautotrophicus RNase P, and can immunoprecipitate RNase P activity when affinity purified against the recombinant proteins, demonstrating that all four proteins are subunits of archaeal RNase P. Yeast two hybrid analyses have demonstrated in vivo interactions between MTH688 and MTH687, which corresponds to findings with both human and yeast nuclear RNase P proteins in similar two-hybrid tests, and between MTH1618 and MTH11, which bb correspond to similar findings with yeast nuclear RNase P subunits. These results demonstrate that the archaeal RNase P holoenzyme has protein subunits homologous to eukaryotic nuclear RNase P, and also suggest that the overall architecture of the enzymes are likely to be similar. ARCHAEAL RIBONUCLEASE P HAS MULTIPLE PROTEIN SUBUNITS HOMOLOGOUS TO EUKARYOTIC NUCLEAR RIBONUCLEASE P SUBUNITS