Figure 6.

Degradation of nascent RNA by Rat1 and Xrn1 is not sufficient to cause pol II termination. (A) Anti-pol II RIP detects nascent transcripts. The wild-type strain (DBY548) was analyzed without (−) and with (+) galactose (gal) induction. Immunoprecipitated RNA was analyzed by RT–PCR with primers for GAL1-ADH4 (position 1) and constitutively expressed TEF1. RT(+) and RT(−) signify plus and minus RT, respectively. (B) Nascent RNA is degraded downstream of the poly(A) site. Anti-pol II RIP of GAL1-ADH4 in galactose-induced wild-type (WT), rat1-1, and rat1-1Δxrn1 strains (DBY548, DBY745, and DBY772) at 25°C or 37°C. Lane 7 is a control − RT. Note the absence of nascent RNA associated with pol II that has failed to terminate at position 4 in lanes 3–5 and stabilization of nascent RNA when both Rat1 and Xrn1 were eliminated (lane 6). This result is representative of four independent experiments. PCR product 4 has 65% as many ³²P-dC residues as product 1. (C) Anti-pol II ChIP of the samples in B. (Lanes 4–6) Note pol II that failed to terminate is present at high density at position 4, although nascent RNA was not detectable in B.