Analysis of RNase P Protein ( rnpA ) Expression in Bacillus subtilis Utilizing Strains with Suppressible rnpA Expression

Abstract

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.Present knowledge about the in vivo function of the bacterial RNase P protein (termed P protein) originates from studies of the Escherichia coli system with a mutant strain (NHY322 [rnpA49]) encoding a P protein with a single amino acid mutation, which results in temperature-sensitive bacterial growth due to decreased affinity of the protein for its RNA subunit (1,7,11,13). Although the rnpA49 mutant's phenotype has indicated that the P protein is essential in E. coli, one caveat is that complementation studies were performed under stress conditions at 43°C. The heat shock response has a global effect on gene expression (14,19), and induced metabolic changes could exacerbate the mutant phenotype. Moreover, the mutant P protein is still expressed in the rnpA49 mutant strain under nonpermissive conditions and may interfere with the function of the P protein expressed from a plasmid in complementation studies.Here we describe the construction and biological properties of Bacillus subtilis mutant strains with suppressible rnpA expression. In one type of mutant strain, termed d7, chromosomal rnpA expression could essentially be abolished, resulting in cell growth arrest. Expression of plasmid-borne rnpA genes encoding either the wild-type (WT) P protein or variants carrying an N-or C-terminal His tag was able to rescue the conditionally lethal phenotype. In contrast, an rnpA gene with internal stop codons was unable to restore growth under restrictive conditions. Our results demonstrate directly that the bacterial P protein is essential for cell viability. We further demonstrate the utility of the d7 strain for RNase P overproduction and affinity purification via His-tagged protein variants. Finally, we show that rnpA expression in B. subtilis is attenuated by transcriptional polarity and that translation of rnpA mRNA in B. subtilis can be initi...

Of proteins and RNA: The RNase P/MRP family

“…The protein component of the bacterial RNase P holoenzyme, a small basic protein, is required for the catalytic activity at physiological conditions and is essential for the survival of the bacterial cell (Schedl and Primakoff 1973;Kole et al 1980;Guerrier-Takada et al 1983;Kirsebom et al 1988;Reich et al 1988;Gossringer et al 2006, and references therein).…”

Ribonucleases P/MRP and the Expanding Ribonucleoprotein World

“…Although RNase P is an essential endonuclease (17)(18)(19) best known for its role in tRNA 5 0 leader maturation (Table 1), to date, several other substrates are known to be processed by this complex (Table 1). Universally, the RNA subunit is able to independently perform catalysis (49)(50)(51) although its enzymatic activity is greatly enhanced in the presence of the protein subunits.…”

The RNR motif of B. subtilis RNase P protein interacts with both PRNA and pre-tRNA to stabilize an active conformer

“…1A). Although the RNA component alone is capable of catalyzing pre-tRNA cleavage in high salt in vitro, the protein is essential for catalytic activity in vivo (Hsieh et al 2004;Evans et al 2006;Gossringer et al 2006;Smith et al 2007). In vitro studies indicate that the P protein subunit contributes to numerous facets of RNase P catalysis including substrate and metal affinity and cleavage efficiency (Reich et al 1988;Crary et al 1998;Kurz et al 1998;Sun et al 2006).…”