In vivo-Analysen zur Funktion bakterieller RNase P-Proteine in Bacillus subtilis

RNase P is responsible for the maturation of tRNA 5Ž termini. The bacterial endoribonuclease is composed of a catalytic RNA and a protein subunit. For complementation studies a Bacillus subtilis strain was constructed that expresses the protein subunit of the RNase P enzyme conditionally. For this purpose the chromosomal rnpA gene (encoding the RNase P protein subunit) was placed under the control of a xylose-promotor (Pxyl) by homologous recombination. RT-PCR analysis demonstrated that expression of rnpA was repressed in the constructed mutant strain efficiently and could be induced in the presence of xylose. In the absence of xylose, a plasmid-encoded homologous rnpA gene could rescue the lethal phenotype showing that the rnpA gene is essential in B. subtilis. Utilizing this strain, a heterologous complementation screen was initiated to compare the functionality of RNase P proteins from phylogenetically distant bacteria in vivo. The conditionally lethal phenotype of the constructed B. subtilis strain could be suppressed by plasmid-encoded RNase P proteins from Escherichia coli and Synechocystis sp. PCC6803. Furthermore, the mutant strain could be exploited to express plasmid-encoded His-tagged RNase P proteins, which provides a method for the isolation of in vivo assembled RNase P holoenzyme by affinity chromatography. Transformation of the mutant strain with an expression vector harbouring rnpA and rnpB (encoding the RNase P RNA subunit) enabled the over production of RNase P holoenzyme.