FIGURE 5.

VapC-mt4-mediated sequence-specific MS2 RNA cleavage. A–K, primer extension analysis for MS2 RNA identified 12 VapC-mt4 cleavage sites. Lanes labeled “−” represent control reactions to which no proteins were added. In lanes labeled “B,” purified His₆-VapB-mt4 was incubated with the MS2 RNA. In lanes labeled “C,” the addition of His₆-VapC-mt4 resulted in the cleavage of the MS2 RNA. In lanes labeled “BC,” preincubation of His₆-VapC-mt4 with His₆-VapB-mt4 prior to the addition of the MS2 RNA prevented RNA cleavage. Cleavage sites/products are indicated by black arrowheads on the right side of gels and in the relevant RNA sequences below each gel. Apparent secondary structure is indicated by white arrowheads on the right side of gels. Lanes G, A, T, and C correspond to DNA sequencing ladders prepared by reverse transcription using the same primers used in the primer extension reactions. Data shown are representative of two independent experiments. L, alignment of sequences containing VapC-mt4 cleavage sites. The ACGC consensus sequence is in bold.