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The principle task of the ubiquitous enzyme RNase P is the generation of mature tRNA 5'-ends by removing precursor sequences from tRNA primary transcripts (Trends Genet 19:561-569, 2003; Crit Rev Biochem Mol Biol 41:77-102, 2006; Trends Biochem Sci 31:333-341, 2006). In Bacteria, RNase P is a ribonucleoprotein composed of two essential subunits: a catalytic RNA subunit (P RNA; 350-400 nt) and a single small protein cofactor (P protein; ∼14 kDa). In vitro, bacterial P RNA can catalyze tRNA maturation in the absence of the protein cofactor at elevated concentrations of mono- and divalent cations (Cell 35:849-857, 1983). Thus, bacterial P RNA is a trans-acting multiple-turnover ribozyme.Here we provide protocols for 5'-endonucleolytic ptRNA cleavage by bacterial P RNAs in the absence of any protein cofactor and under single-turnover conditions ([E] >> [S]). Furthermore, we outline a concept that utilizes the bacterial RNase P ribozyme to release RNAs of interest with homogeneous 3'-OH ends from primary transcripts via site-specific cleavage. Also, T7 transcription of mature tRNAs with clustered G residues at the 5'-end may result in 5'-end heterogeneities, which can be avoided by first transcribing the 5'-precursor tRNA (ptRNA) followed by P RNA-catalyzed processing to release the mature tRNA carrying a homogeneous 5'-monophosphate end. Finally, RNase P ribozyme activity can be directly assayed by using total bacterial RNA extracts.