FIGURE 7.

In vitro analysis of the cleavage of model substrates representing the Rnt1p cleavage site in the 3′-ETS or site A4, respectively. (A) Structural comparison of the model substrates. Conserved nucleotides are boxed. The shaded box highlights the nucleotides in the ITS1 stem that are strongly conserved among various yeast strains (Eppens et al. 2002). (B) Transcripts corresponding to the model substrates were prepared by in vitro transcription, 5′-end labeled, and tested for cleavage by incubating them with 0.8 pmol of recombinant Rnt1p in the presence of two different concentrations of KCl. Cleavage products were fractionated on 20% denaturing polyacrylamide gels. The RNA molecular weight marker is shown on the left. The positions of the substrate (S) and product (P) are indicated.