Analysis of RNase P protein (rnpA) expression in Bacillus subtilis utilizing strains with suppressible rnpA expression.

Bacterial RNase P is composed of an RNA subunit and a single protein subunit (encoded by the rnpB and rnpA genes, respectively). We constructed Bacillus subtilis mutant strains that conditionally express the RNase P protein under control of the xylose promoter (P(xyl)). In one strain (d7), rnpA expression was efficiently repressed in the absence of the inducer xylose, leading to cell growth arrest. Growth could be restored by a second functional rnpA allele. This is the first RNase P protein knockdown strain, providing the first direct proof that the rnpA gene is essential in B. subtilis and, by inference, in other bacteria. We further show (i) that, in the wild-type context, rnpA expression is attenuated by transcriptional polarity and (ii) that translation of rnpA mRNA in B. subtilis can be initiated at two alternative start codons. His-tagged RNase P protein variants are functional in vivo and permit purification of in vivo-assembled holoenzymes by affinity chromatography. Simultaneous expression of plasmid-encoded RNase P RNA and His-tagged protein increased RNase P holoenzyme yields. Massive overproduction of RNase P protein in strain d7 is compatible with cell viability.

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