FIGURE 7.

Cleavage of the E. coli tRNA^Tyr precursor substrate by the Arabidopsis and E. coli enzymes. (A) Arabidopsis and E. coli enzymes were incubated with a 131-nt [³²P]-labeled RNA corresponding to the precursor of E. coli tRNA^Tyr for 0, 5, 15, and 45 min. The RNAs harbor either one (5′-P) or three (5′-PPP) phosphates at the 5′ end as indicated at the top. Following incubation, RNA was purified and analyzed on denaturing PAGE and autoradiography. The substrate (S) as well as the 3′ and 5′ cleavage products are indicated at the left. (B) A schematic representation of the 131-nt E. coli tRNA^Tyr precursor is adapted from Soderbom et al. (2005). The RNase P canonical cleavage site is indicated (M1). Arabidopsis RNase E cleavage sites, as mapped in the experiments described in Figures 7 and 8, are shown with arrows. Three guanidine residues added to the 5′ end of the RNA substrate are enclosed in a dashed box. (C) Cleavage of the tRNA^Tyr precursor transcript by the M1 RNA of E. coli RNase P. Conditions are as in A. (D) Inhibition of cleavage activity by EDTA treatment and the inactive mutant of Arabidopsis RNase E (K→A). Incubation times were 5, 15, and 45 min. The substrate is the tRNA^Tyr precursor transcript. EDTA treatment was performed by addition to a final concentration of 10 mM. Other conditions are as in A. Ar, Arabidopsis RNase E. (E) Arabidopsis and E. coli enzymes were incubated with the tRNA^Tyr precursor for 30 min in the presence of Mg²⁺ as indicated. Following incubation, the RNA was purified and analyzed by denaturing PAGE and autoradiography. The 5′ cleavage product is not shown in this figure.