Figure 3. Analysis of transcription termination at ARS305.

(A) Scheme of the reporter system (Porrua et al., 2012) used to assess termination at ARS305. P_TETOFF: doxycycline-repressible promoter; P_GAL: GAL1 promoter. Termination of transcription at a candidate sequence (blue) allows growth on copper containing plates while readthrough transcription inhibits the GAL1 promoter and leads to copper sensitivity, as indicated. (B) Growth assay of yeasts bearing reporters containing a Reb1-dependent terminator, (Colin et al., 2014, used as a positive control), or ARS305 (lanes 1 and 3, respectively). Variants containing mutations in the Reb1 binding site (Reb1 BS ‘−') or the ACS sequence are spotted for comparison (lanes 2 and 4, respectively). (C) Northern blot analysis of P_TET transcripts produced in wild-type and rrp6∆ cells from reporters containing either a Reb1-binding site (Reb1 BS, lanes 1–2) or wild-type or mutant ARS305 sequences, as indicated (lanes 3–8). Transcripts terminated within ARS305 or at the CUP1 terminator are highlighted.

Figure 3—figure supplement 1. ARS305 sequence confers mitotic maintenance to a centromeric plasmid when transcription is shut down.

To assess the functionality of ARS305 in the reporter construct used for detecting transcription termination, we deleted the 2µ origin of the plasmid and transformed yeast in the presence or absence of doxycycline to control expression of the TET promoter. Transformants were only recovered in the absence of transcription, indicating that ARS305 is active but inactivated, as expected, when strong transcription runs through it. Candelli et al., Table 1.