The gene coding for H1 RNA, the RNA component of human RNase P, has been isolated and characterized from a human genomic DNA library. The sequence corresponding to the mature H1 RNA is almost identical to that previously identified using H1 RNA and a cDNA clone corresponding to it. The nucleotide sequence of the genomic clone contains an array of potential transcriptional control elements, some characteristic of transcription by RNA polymerase III and some characteristic of RNA polymerase II, as is also the case for U6 and certain other small stable RNAs. The transcription in vitro of the genomic clone shows that the gene is functional and is transcribed by RNA polymerase III. Southern hybridization analysis indicates that there is very likely only one copy of the gene for H1 RNA in the human genome.

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We have studied the assembly of Escherichia coil RNase P from its catalytic RNA subunit (Ml RNA) RNase P is an endoribonuclease which cleaves the 5' ends of precursor tRNA molecules to create their mature 5' ends. In Escherichia coli, it consists of a protein component, C5 (molecular weight, 13,800), and an RNA component, Ml (377 nucleotides in length) (1, lb). The catalytic activity of the enzyme resides in the RNA (5). Ml RNA alone can carry out the cleavage reaction in vitro, in buffers containing >20 mM Mg2+. In vivo and in buffers containing 10 mM Mg2+, both Ml RNA and C5 protein are required for the RNase P reaction to proceed (15,17). C5 protein increases the kcat of the reaction catalyzed by Ml RNA 10-to 20-fold (1, lb, 13) and also plays a role in the recognition of specific tRNA precursor substrates (9).The rnpA49 mutation in the rnpA gene, the gene coding for C5 protein in E. coli (17), results in an arginine-to-histidine alteration at position 46 in the C5 protein (9). Cells containing this mutation do not grow above 42°C on LB plates and show an accumulation of precursor tRNAs when shifted from a permissive to a nonpermissive temperature (18). Even under permissive growth conditions, cells bearing the rnpA49 mutation show a modest accumulation of certain precursor tRNAs (18) and a reduced (though not zero) production of some mature tRNAs (8,14,18). Interestingly, cells bearing the rnpA49 mutation will grow above 42°C when they contain a plasmid carrying the rnpB gene, which codes for Ml RNA (8,11,14). Presumably, complementation of the temperature-sensitive phenotype is related to the production of excess Ml RNA in the cells containing the plasmid (8,14). It has been suggested that an increase in the efficiency of assembly of the holoenzyme itself is directly responsible for the complementation (11). To explore the validity of this explanation of the complementation phenomenon, we have studied aspects of the assembly in vitro of RNase P from its separated RNA and protein subunits.Although the rnpA49 mutation was first reported in 1973 (17), the RNase P holoenzyme containing C5 protein with the rnpA49 mutation has been studied only in vitro with * Corresponding author.crude cell extracts or partially purified protein preparations (10, 18), both of which yield very little enzymatic activity. Additionally, while the enzymatic activity in crude extracts of rnpA49 was temperature sensitive, no such behavior was observed with RNase P reconstituted with partially purified C5A49 protein and Ml RNA (10). There is one report of temperature-dependent inactivation of RNase P partially purified from rnpA49 (6), but no direct comparison of similar inactivation studies of wild-type RNase P was provided.We have now cloned the rnpA49 gene in a manner which allows the production of large quantities of C5A49 protein.We have purified this protein to apparent homogeneity and have studied the activities in vitro of the RNase P holoenzyme formed from this protein and of Ml RNA made by transcription in vitro. We show tha...