The RNA polymerase II CTD is essential for 3' end cleavage of metazoan pre-mRNAs and binds 3' end processing factors in vitro. We show genetic and biochemical interactions between the CTD and the Pcf11 subunit of the yeast cleavage/polyadenylation factor, CFIA. In vitro binding to Pcf11 required phosphorylation of the CTD on Ser2 in the YSPTSPS heptad repeats. Deletion of the yeast CTD reduced the efficiency of cleavage at poly(A) sites, and the length of poly(A) tails suggesting that it helps couple 3' end formation with transcription. Consistent with this model, the 3' end processing factors CFIA, CFIB, and PFI were recruited to genes progressively, starting at the 5' end, in a process that required ongoing transcription.
We investigated whether the multisubunit holoenzyme complex of RNA polymerase II (Pol II) and mediator is universally required for transcription in budding yeast. ⌬CTD Pol II lacking the carboxy-terminal domain of the large subunit cannot assemble with mediator but can still transcribe the CUP1 gene. CUP1 transcripts made by ⌬CTD Pol II initiated correctly and some extended past the normal poly(A) site yielding a novel dicistronic mRNA. Most CUP1 transcripts made by ⌬CTD Pol II were degraded but could be stabilized by deletion of the XRN1 gene. Unlike other genes, transcription of CUP1 and HSP82 also persisted after inactivation of the CTD kinase Kin28 or the mediator subunit Srb4. The upstream-activating sequence (UAS) of the CUP1 promoter was sufficient to drive Cu 2+ inducible transcription without Srb4 and heat shock inducible transcription without the CTD. We conclude that the Pol II holoenzyme is not essential for all UAS-dependent activated transcription in yeast. RNA polymerase II (Pol II) holoenzymes containing core polymerase and many accessory proteins have been identified in budding yeast (Thompson et al. 1993;Kim et al. 1994;Koleske and Young 1994) and in mammals (Ossipow et al. 1995;Maldonado et al. 1996;Pan et al. 1997). In yeast up to 50% of Pol II is bound to a complex of ∼20 proteins called mediator (Kim et al. 1994;Myers et al. 1998). Addition of mediator to core Pol II in vitro elevates basal transcription and permits stimulation of transcription in response to activators (Kim et al. 1994;Koleske and Young 1994). In addition to the mediator, holoenzyme has also been isolated in association with other proteins including TFIIB, TFIIF, TFIIH, and Swi/ Snf (Koleske and Young 1994;Wilson et al. 1996).Several lines of evidence suggest that the carboxy-terminal domain (CTD) of the Pol II large subunit is essential for the physical and functional integrity of the holoenzyme. Purified mediator binds to the CTD (Myers et al. 1998) and the Pol II-mediator complex is disrupted by a monoclonal antibody against the CTD (Kim et al. 1994). Furthermore, addition of mediator to Pol II lacking the CTD did not stimulate either basal or activated transcription (Myers et al. 1998). The CTD is composed of a repeated heptad sequence whose consensus (YSPTSPS) is absolutely conserved between yeast and mammals. The CTD undergoes a cycle of hyperphosphorylation and dephosphorylation that accompanies the transcription cycle (Dahmus 1996) and may be linked to recycling of the mediator (Svejstrup et al. 1997). Mediator also strongly stimulates CTD phosphorylation by the Kin28 kinase subunit of TFIIH (Kim et al. 1994). It has been suggested that phosphorylation of the CTD counteracts a negative effector of transcription in crude extracts and that it enhances transcriptional elongation (O'Brien et al. 1994;Lee and Greenleaf 1997). Truncation of the yeast CTD from 26 to between 13 and 11 repeats causes cold and temperature sensitivity for growth and further deletion to 10 or fewer repeats is lethal (Nonet et al. 1987b;West a...
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