Figure 7.

The NNS and the CPF pathways have largely overlapping sequence determinants. (A) A scheme of the reporter construct containing the 1.1-kb LEU2 gene inserted between the P_Tet and the P_Gal promoters. Artificial CUT sequences were cloned by recombination downstream of LEU2 as indicated. A stuffer fragment from the HSP104 gene (486 nt) is present after the terminators insertion point as polymerases committed to CPF-dependent termination are known to transcribe downstream of the terminator. (B) Northern blot analysis of RNAs derived from clone 78 and two additional artificial terminators cloned downstream of LEU2. These constructs were expressed in an rna14-3 strain to assess the dependency on the CPF pathway. A Δrrp6 strain was employed to stabilize read-through transcripts generated by mutation of Rna14p. (C) Copper growth assay of cells expressing the constructs containing the artificial terminators cloned downstream of LEU2 as indicated (3′). A scheme indicates the positions of the relevant motifs. Sequences are reported in Figures 3A and 4A. For comparison, clones 78 and 78-INV inserted in a 5′ position (inducing termination by the NNS pathway) are also included in the test set as indicated.