FIGURE 6.

mRNA cleavage is not required for VapC-mt4-mediated translation inhibition. A, purified His₆-VapC-mt4 inhibits coupled in vitro transcription/translation of the PinPoint-CAT fusion protein (lane 3). His₆-VapB-mt4 can rescue the inhibition and reconstitute translation (lane 5). B, schematic representation of the open reading frames encoded in the PinPoint Xa Control vector. The black rectangle denotes the PinPoint peptide. The white and gray arrows correspond to the ORFs encoding the 27.5-kDa CAT protein and the 31.5-kDa β-lactamase, respectively. The 39.5-kDa PinPoint-CAT fusion protein and β-lactamase are the major proteins expressed using the PinPoint Xa Control vector. The bent arrow shows the location of the T7 promoter. Thin arrows correspond to primers used for RT-PCR. C, the RNA was extracted from the coupled in vitro transcription/translation system, and RT-PCR was performed to assess the state of the mRNA encoding the PinPoint-CAT fusion protein. The mRNA encoding the PinPoint-CAT fusion protein was not cleaved by His₆-VapC-mt4. Data shown are representative of two independent experiments.