Figure 1.
Synthesis of a novel CUP1 transcript by ΔCTD Pol II. (A) Northern blots of mRNA from rpb1-1ts strains at intervals after shifting to the restrictive temperature of 37°C. Strain Y260 was transformed with pFL38 vector (lanes 1–5), pFL38–RPB1 (lanes 6–10), or pFL38–rpb1ΔCTD (lanes 11–15). Note the appearance of a new 1.7-kb CUP1 transcript specific to the rpb1ΔCTD strain. (B) 5′ and 3′ end mapping of CUP1 transcripts. RNase protection analysis with antisense probes A (lanes 1,2), B (lanes 3,4), and G (lanes 5,6; see D) of RNA from Y260 containing pFL38 grown at 30°C (vector; lanes 1,3,5) or pFL38–rpb1ΔCTD grown for 1 hr at 37°C (ΔCTD; lanes 2,4,6). Note that CUP1 RNAs have identical 5′ ends in the two samples (arrowhead; lanes 1,2), but the ΔCTD Pol II-expressing cells have transcripts that extend past the normal poly(A) site (arrowheads; lane 4). Multiple bands in lane 3 are attributable in part to artifactual cleavage at AU rich regions in probe B. The 3′ ends of the readthrough RNAs map to the end of the YHR54C ORF (arrowhead; lane 6). Markers (M, lane 7) are MspI cut pBR322 from 427–123 bp. (C) 5′ end mapping of hybrid selected transcripts from the CUP1 locus in pFL38–rpb1ΔCTD cells grown at 30° (t = 0, lanes 1–3) and for 60 min at 37°C (t = 60; lanes 4–6). Total RNA (T; lanes 1,4) and RNA hybrid selected with a 5S rDNA and either CUP1 A or G (see D) were analyzed by RNase protection with probes for 5S rRNA and the CUP1 5′ end (probe A). Note that only at 37°C (lane 6) did transcripts homologous to region G appear and their 5′ ends were identical to those of normal CUP1 transcripts made at 30°C (lanes 1,2). (D) Map of the CUP1 repeat unit with 0.6- and 1.7-kb transcripts and fragments A–G indicated.