Cyanobacterial RNase P recognition of the FMDV IRES. (A) Ribozyme concentration-dependent cleavage of the FMDV IRES by the Synechocystis RNase P. A constant concentration of uniformly labeled FMDV IRES (6.75 nM) was incubated with increasing concentrations of Rz 6803; the digestion products were fractionated on 6% denaturing acrylamide gels and visualized by autoradiography. P1–P4 depict the most intense specific digestion products, absent in the control RNA lane incubated in the absence of ribozyme. (B) Influence of the Mg2+ concentration in the cleavage efficiency of the FMDV IRES by the Synechocystis RNase P. Uniformly labeled FMDV IRES (6.75 nM) was incubated with Rz 6803 in a buffer containing the indicated concentration of Mg2+; the digestion products were fractionated on 6% denaturing acrylamide gels and visualized on X-ray films. (C) Determination of the average products length. The digestion products obtained in reactions similar to those described in A and B were fractionated in long denaturing gels in parallel to known labeled transcripts indicated to the left of the autoradiogram, used as markers. The substrate concentration in lanes 2, 3, and 4 was 67.5–6.75 nM with a constant concentration of ribozyme (67.5 nM).
