The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase-CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.Eukaryotic pre-mRNAs are transcribed by RNA polymerase II (Pol II) and undergo several processing events before maturing into mRNA. Soon after initiation of transcription, premRNA is capped at its 5Ј terminus (27,48). Transcripts are further processed by the splicing and polyadenylation machineries before translocation to the cytoplasm for translation. Cotranscriptional mRNA processing is facilitated by the recruitment of mRNA processing factors to the carboxy-terminal domain (CTD) of the Pol II large subunit (8,22,23,26,36,37,56).The CTD is composed of a tandemly repeated heptad with the consensus sequence YSPTSPS (1, 10). Mammalian Pol II CTD has 52 repeats, whereas the yeast Saccharomyces cerevisiae CTD has only 26 (12). Deletion of the mouse (4), Drosophila (58), or yeast (2, 43) CTD is lethal, and partial deletions result in conditional phenotypes, reducing transcription and response to activators (5,19,38,49). The CTD is phosphorylated in vivo, primarily at serine 2 and serine 5 of the heptapeptide consensus repeat (12). Hyperphosphorylation of the CTD appears to be coordinated with transcription initiation and elongation in vivo (45, 54). Phosphorylation is mediated by one or more CTD kinase activities, but the timing and role of specific kinases are not clearly defined.Several putative CTD kinases are members of the cyclindependent kinase (CDK) family. These kinases typically consist of a catalytic subunit bound to a regulatory cyclin subunit. The Kin28-Ccl1 (Cdk7-cyclin H) kinase complex associated with the general transcription factor TFIIH can phosphorylate the CTD after preinitiation complex (PIC) formation, thereby positively regulating transcription (16,21). The Srb10-Srb11 (Cdk8-cyclin C) kinase complex is associated with the RNA Pol II h...