When we began to look at RNase P in Archaea, very little was known about them, and most of what was known was contradictory. But it was clear that these enzymes contained an RNA, but these RNAs could not be shown to be catalytically active in the absence of protein. When we began this work, we started by cloning and sequencing the RNase P RNA genes from a variety of halophilic Archaea and the methanogen Methanosarcina barkeri. During this time, the genome sequence of Methanocaldococcus jannaschii became available as well, and we identified the RNase P RNA sequence in this genome. None of these RNAs, transcribed in vitro were by themselves catalytically active, and it became accepted by the field that archaeal RNase P RNAs, like those of eukaryotes, absolutely required their protein(s) for function.
The Haloferax volcanii enzyme had been examined in two published reports, one from the Sid Altman lab and the other from the Chuck Daniels lab. In the Altman paper, the enzyme was described as being nuclease sensitive, and so presumably containing an essential RNA component. Also consistent with the presence of an RNA was its bouyant density of 1.61 in cesium sulfate gradients. It appeared to be larger than the E.coli or human enzymes, at 18S, and functioned optimally at 55mM Mg++ and less than 100mM KCl or NH4Cl. They made a protein extract from the partially-purified H. volcanii RNase P and claimed they could reconstitute RNase P activity with this protein and the E.coli RNase P RNA (the reciprocal experiment, with RNA from the H. volcanii enzyme and the E. coli RNase P protein did not yeild activity). In the Daniels paper, an RNA of 435nt that copurified with the H. volcanii RNase P is identified, cloned, and sequenced. This RNA is recognizably similar to bacterial RNase P RNAs, but in vitro transcripts of this RNA by itself failed to yeild catalytic activity under any conditions tested, although in personal communications Chuck claimed that the RNA was sporatically active. They went on to show that they could add the RNase P protein from B. subtilis to this RNA and reconstitute RNase P activity. They also showed that the H. volcanii enzyme pellets rapidly through glycerol gradients (consistant with a large complex), was nuclease sensitive, and in their hands was inactivated by concentrations of cesium sulfate required for density gradient centrifugation.
The Sulfolobus acidocaldarius RNase P enzyme was described in a pair of papers from the Norm Pace lab. In the initial report, the enzyme was reported to function optimally at 77C in ca. 50mM ammonium and 10mM Mg++, and an apparent size of ca. 400 kDa. The density of the enzyme in cesium sulfate was 1.27, essentially the same as protein alone, and the activity of the enzyme was resistant to micrococcal nuclease, both implying the absence of RNA! Nevertheless, the enzyme does contain copurifying RNA that is not degraded during nuclease treatment. In the subsequent paper, the RNA is identified, cloned, and sequenced; it is a bona fide RNase P RNA. This RNA, like that of H. volcanii, was not by itself active under any conditions tested. The addition of bacterial RNase P protein failed to reconstitute activity from the RNA, and they were unable to repeat the reconstitution with the H. volcanii RNA described by the Daniels lab. The paper also presents a preliminary model for the secondary structure of the RNA based on comparative analysis.