Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II plays an important role in transcription and processing of the nascent transcript by interacting with both transcription and RNA processing factor s. We show here that the cleavage/polyadenylation factor IA of Saccharomyce s cerevisiae directly contacts CTD, First by affinity chromatography experi ments with yeast extracts we demonstrate that the Rna15p, Rna14p, and Pcf11 p subunits of this complex are associated with phosphorylated CTD, This int eraction is confirmed for Rna15p by yeast two-hybrid analysis. Second, Pcf1 1p, but not Rna15p, is shown to directly contact phosphorylated CTD based o n in vitro binding studies with recombinant proteins. These findings establ ish a direct interaction of cleavage/polyadenylation factor IA with the CTD . Furthermore, a quantitative analysis of transcription run-on performed on temperature-sensitive mutant strains reveals that the lack of either funct ional Rna14p or Pcf11p affects transcription termination more severely than the absence of a functional Rna15p, Moreover, these data reinforce the con cept that CTD phosphorylation acts as a regulatory mechanism in the maturat ion of the primary transcript.