Mitochondrial ribonuclease P activity of Trypanosoma brucei

Ribonuclease P (RNase P) is an essential enzyme that cleaves the 5 ' leader sequences of precursor tRNAs (pre-tRNAs) to generate mature tRNAs. The RNa se P-like activity from Trypanosoma brucei mitochondria (mtRNase P) was pur ified over 10000-fold by sequential column chromatography. This is the firs t demonstration of such activity from mitochondria of parasitic protozoa. I ts apparent molecular weight is similar to 70 kDa., considerably less than bacterial RNase P. Preliminary characterizations revealed no RNA component that is essential for this activity. Like other RNase P activities, the cle avage generates mature tRNAs with a terminal 5 ' -phosphate at the cleavage site and the 5 ' leader sequence with a 3 ' -hydroxyl. Disruption of the p re-tRNA tertiary structure inhibits the cleavage of the substrates. These d ata suggest that although all mitochondrial tRNAs are encoded in nuclear DN A in T. brucei, these cells contain an RNase P in the mitochondrion that cl eaves the 5 ' terminal leader sequences of pre-tRNAs to generate mature tRN As. Cleavage by mtRNase P of a pre-tRNA substrate that was divided into two fragments was demonstrated. This shows the feasibility of artificial regul ation of gene expression that can be achieved by creating a complex made of target mRNA and a complementary small oligonucleotide that resembles natur al substrates for RNase P. (C) 2001 Elsevier Science B.V. All rights reserv ed.