Fig. 5.

vhs cleaves truncated transcripts containing the IEX-1 coding domains at multiple sites. (A) Schematic representation of the full-length IEX-1 mRNA and location of the primers used to generate the DNA template for in vitro transcription of IEX-1 coding domain (substrate 3). (B) Five micrograms of either GST-vhs fusion protein (lanes 2–5) or GST (lanes 6–9) were incubated at 30°C with internally labeled substrate 3. Samples were removed at the indicated times, purified, and resolved on a sequencing gel, followed by autoradiography as described in Materials and Methods. Internally labeled RNA marker was loaded in lane 1, and the length of the fragments is reported on the side. (C) Internally labeled substrate 3 was incubated with different amounts of GST-vhs fusion protein: 4 μg (lanes 1–3), 2 μg (lanes 4–6), and 1 μg (lanes 7–9). Samples were analyzed as in B. (D) 5′ end-labeled substrate 3 was incubated with 5 μg of purified GST-vhs fusion protein at 30°C for the indicated times. Samples were analyzed as in B and C.