Fig. 4.

Determination of RNase P minimal substrate and cleavage specificity in the tRNA-mimic region 1. To delimit the human RNase P minimal substrates in IFNA5 mRNA, shortened DNA templates were obtained by PCR, T7 in vitro transcribed in the presence [α-³²P]GTP and subjected to cleavage. a Schematic representation of shortened IFNA5 RNA transcripts (197–446), (215–427) and (329–427). b Human RNase P activity test under standard conditions. The three fragments were incubated on ice (lanes 2, 7 and 12), in reaction buffer (lanes 3, 8 and 13), or in the presence of human RNase P (lanes 4, 9 and 14). Unlabelled pre-tRNA^Tyr (10× molar excess) or poly-r(A) (equivalent quantity by weight) was added to the reaction in lanes 5, 10 and 15 and 6, 11 and 16, respectively. Lanes 1 and 17 are the century and decade molecular markers, respectively. c Cyanobacterial ribozyme dose–response essay. The three fragments were incubated on ice (lanes 1, 6 and 11), in the presence of buffer (lanes 2, 7 and 12) or with bacterial ribozyme at 67.5 nM (lanes 3, 8 and 13), 337.5 nM (4, 9 and 14) and 675 nM (lanes 5, 10 and 15), respectively. Lane 16 contains the century molecular markers