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So, what is RNase P? RNase P is an enzyme in the pathway for transfer RNA biosynthesis. It is the endonuclease that cleaves the pre-tRNA at the junction of the tRNA and the 5' leader, generating the mature 5' end of the tRNA. It is a metalloenzyme, preferring Mg++, and leaves a 5' phosphate on the mature tRNA and a 3' hydroxyl on the leader RNA. In vivo it seems to generally act before the 3' processing of pre-tRNAs, but in vitro it clearly prefers substrates that already have the mature 3'-NCCA end.

The two gel lanes to the right show how we assay this enzymatic activity. The uppermost band is the pre-tRNA exactly as shown to the left (this is a Bacillus subtilis pre-tRNA(Asp) with an arbitrary leader sequence and mature 3' end), uniformly labeled during it's synthesis by in vitro transcription with alpha-32P-GTP. Treatment of this pre-tRNA with RNase P (the "+" lane) results in its cleavage into two products; the upper band is the mature tRNA, the lower is the 5' leader RNA. By measuring the radioactivity in each band, the amount of catalytic activity can be precisely quantitated for enzymological analysis.