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This is an RNase P activity gel, testing RNase P RNAs transcribed in vitro from cloned genes for the ability to cleave pre-tRNA (the Asp tRNA of B. subtilis). The first lane shows the size of the substrate, which can be cleaved to yeild the mature length tRNA and the leader RNA by the E. coli RNase P RNA alone. As you can see, several of the archaeal RNase P RNAs also had the ability, in the absence of protein, to cleave this substrate. All those of the methanobacteria (M.formicicum, M.thermoautotrophicum, M.thermoautotrophicus, and isolates ER-H and ER-E) had the best activity; these are the RNAs that most closely resemble those of Bacteria. Those of the thermococci (Thermococcus celer and Pyrococcus furiosus) also had detectable activity, as did those of the extreme halophile H. volcanii and, on the limits of detection, the alkylhalophile Natronobacterium gregoryi.

Why hadn't we seem this catalytic activity by the archaeal RNAs before? Partly this was bad luck; the first RNAs available, other than that of H. volcanii were among those that lack activity. The other reasons are the conditions required for activity and the levels of activity. This activity requires very high ionic strength (4M ammonium!) and Mg++ (300mM). And these assays contain a 200-fold excess of enzyme, and are incubated for hours!

Given the contradictory work from the past, and the low levels of activity, it was important to show that this activity was not from traces of contamination from external sources. This was checked by several approaches, but the most interesting are that a uni-molecular RNase P:pre-tRNA cleaves in cis, and total cellular RNAs from these same organisms also contain RNase P acitivty under these conditions.

Despite their poor activity, the ability of these RNAs to cleave precursors in the absence of protein at any rate implies that, like those of Bacteria, they contain everything necessary for both substrate recognition and catalysis. Further characterization of this RNA-alone activity in M. thermoautotrophicus showed that the RNA is structurally deranged (Pb++ sensitive, temperature sensitive), consistent with the requirement for high ionic stregnth for activity. The affinity for substrate (Km) is very low, at least 1000-fold higher than the bacterial RNA, but there is no apparent defect in catalysis itself (kcat).

We can also reproduce Chuck Daniels "unreproducable" activity by the Haloferax RNA. This RNA, it turns out, is active (minimally) in 4M ammonium, but not 3M. His assays were in 3M ammonium acetate. These reactions, when incubated in an regular incubator, fail to show catalytic activity. But when incubated in a heating block, evaporation of the sample over the time of incubation and accumulation of the reaction water on the lid of the tube results in elevation of the ionic strength of the reaction itself, and these reaction tubes show catalytic activity!