Phosphorylation Cycle of the Pol II CTD

During the transcription cycle, the CTD of RNA Pol II undergoes a cycle of phosphorylation and dephosphorylation (Dahmus, 1996). Hypophosphorylated Pol II is the form that is recruited to promoters for formation of the pre-initiation complex. Shortly after transcription begins, the CTD becomes heavily phosphorylated (Lin et al., 2002). In eukaryotes, three major cyclin-dependent kinases, CDK7, CDK8, and CDK9 specifically target and phosphorylate the CTD of RNA Pol II (Prelich, 2002). Although these three kinases are part of different protein complexes, they themselves are evolutionarily conserved in all eukaryotes. CDK7 (also called MO 15 or Kin28 in yeast) is a component of the general transcription factor TFIIH, which contains several core subunits and an enzymatically active CAK sub-complex consisting of CDK7, cyclin H and Matl (Orphanides et al., 1996). Whereas the TFIIH-free CAK can exist as a free, independent complex and function as a cell cycle regulator by targeting and activating a set of CDKs that are involved in cell cycle progression, the association with the TFIIH core subunits changes CAK's substrate specificity and enables it to phosphorylate the CTD at the Ser5 position within the heptapeptide repeats (Akoulitchev et al., 1995). The phosphorylation of Ser5 is not only essential for the formation of the first phosphodiester bond in the nascent RNA chain, but also important for Pol II to clear the promoter and shift into an early elongation mode (Orphanides and Reinberg, 2002; Rodriguez et al., 2000; Sims et al, 2004). Moreover, it also allows the CTD to recruit capping activities to the 5' end of the nascent pre-mRNA (also see below) (Komarnitsky et al, 2000; Rodriguez et al, 2000; Schroeder et al, 2000).

The second CTD kinase, CDK8, is a component of the Mediator complex, which plays an intermediary role in bridging the interactions of transcriptional activators with the basal transcriptional apparatus. CDK8 has been reported to function in transcriptional events prior to the elongation step owing to its ability to phosphorylate the Pol II CTD (Leclerc et al, 1996; Nelson et al, 2003; Rickert et al, 1996). However, its precise role in regulating transcription remains largely unknown.

Soon after transcription initiation, the transcription elongation complex is arrested at a "checkpoint" (Fig. 14.1) to ensure proper pre-mRNA capping. Release from this checkpoint requires the enzymatic action of the third CTD kinase CDK9 (Burl or Ctkl in yeast), which targets Ser2 in the CTD. From now on till the end of the transcription cycle, phosphorylation of Ser2 predominates (Price, 2000; Zhu et al., 1997). This particular phosphorylation event enables Pol II to resist pausing caused by the concerted actions of DSIF and NELF (see below for details) (Wada et al., 1998; Yamaguchi et al., 1999; Yamaguchi et al., 2002). The phosphorylation of Ser2 in the CTD, the Spt5 subunit of DSIF (Ivanov et al. 2000) and the RD subunit of NELF (Fujinaga et al. 2004) by CDK9 results in heavily phosphorylated Pol IIo, converts DSIF and NELF into positive elongation factors, and enables a shift from the abortive to the productive phase of transcriptional elongation (Renner et al., 2001; Wada et al., 1998; Yamaguchi et al., 1999). In addition, the Ser2 phosphorylation functions in part by recruiting RNA processing factors involved in pre-mRNA splicing and 3' end polyadenylation to facilitate the cotranscriptional processing (see below for details).

At the end of the transcription cycle and upon the generation of a mature mRNA, Pol II must dissociate from DNA and undergo dephosphorylation in order to be recycled into the hypophosphorylated Ila form for subsequent rounds of transcription (Cho et al., 2001; Licciardo et al., 2001; Palancade et al., 2001). A protein phosphatase named FCP1 is the best-characterized enzyme that acts on the Pol II CTD (Fig. 14.1). Human FCP1 is able to dephosphorylate both Ser2 and Ser5 in the CTD, whereas the S. pombe FCP1 displays a preference for Ser2 over Ser5 (Cho et al., 2001; Hausmann et al., 2004; Mandal et al., 2002). Interestingly, besides participating in the recycling of Pol II, FCP1 was found to associate with the elongating Pol II both in vivo and in vitro and exhibit elongation stimulatory activity in a manner independent of its phosphatase catalytic activity (Cho et al., 2001; Mandal et al., 2002). In addition to FCP1, a few other protein phosphatases (e.g. Ssu72 and protein phosphatase 1) have also been shown to target the CTD and implicated in recycling of the RNA Pol II (Krishnamurthy et al., 2004; Washington et al, 2002), although the mechanistic details of their actions remain very sketchy at this moment.

PIC assembly





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