Figure 4.

In vitro cleavage of tRNAs^Arg by colicin D. The purified colicin D (2–50 nM) was preincubated with or without 100–500 nM ImmD for 15 min at 37°C in 10 mM Hepes⋅KOH (pH 7.8) and 1 mM DTT. E. coli tRNA mixture (derived from MRE 600; Sigma) was then added to the reaction mixture to 5.0 A₂₆₀ units/ml, followed by incubation for 10 min at 37°C. The RNAs were separated by electrophoresis on a 10% polyacrylamide gel containing 7 M urea, and then were analyzed by Northern blot hybridization using DNA probes specific to tRNA^ArgICG (A) and tRNA^ArgCCG (B). The arrows indicate cleaved 3′ fragments of the tRNAs^Arg. The weaker signal for the uncleaved tRNA^ArgCCG band compared with that for the cleaved one is possibly attributable to different efficiencies of hybridization and/or transfer to the membrane.