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Fig.2.3 Purification of human RNA polymerase II complexes. Four different forms of RNA polymerase II (pol II) are purified from a stable cell line (hRPB9-3) conditionally expressing FLAG epitope-tagged human RPB9 (Wu and Chiang, 2001b). Pol II holoenzyme can be purified from the cytoplasmic SI00 or nuclear extract fraction. Similarly, the IIA (i.e., containing hypo-or unphosphorylated CTD) form of core pol II comprised of RPB1 to RPB12 subunits can also be isolated from the same S100 or nuclear extract fraction, but under high salt wash conditions (Kershnar et al„ 1998). The IIO (i.e., containing hyper-phosphorylated CTD) and the IIB (i.e., CTD-truncated) forms of pol II can be additionally purified from the nuclear pellet. The tail represents the CTD (carboxy-terminal domain) found in the RPB1 subunitofpol II.

Protein phosphatases that remove the phosphate group on serine 2 or serine 5 have also been identified. Yeast Ssu72 (Krishnamurthy et al., 2004), plant Arabidopsis thaliana CTD phosphatase-like proteins AtCPLl and AtCPL2 (Koiwa et al., 2004), and human small CTD phosphatase 1 (SCP1) protein (Yeo et al., 2003) are able to dephosphorylate serine 5, whereas TFIlF-associated CTD phosphatase 1 (Fcpl) isolated from yeast (Archambault et al., 1997; Kimura et al., 2002) and humans (Archambault et al., 1998; Cho et al., 1999) are mainly implicated in serine 2 dephosphorylation (Cho et al., 2001). Fcpl interacts with TFIIB (Chambers et al., 1995; Kobor et al., 2000), the RPB4 subunit of pol II (Kimura et al, 2002), and the RAP74 component of TFIIF (Chambers et al, 1995; Kobor et al., 2000). It has been shown that TFIIF can stimulate Fcpl phosphatase activity and may thus accelerate reinitiation of transcription by enhancing the conversion of pol II from the elongating IIO form back to the initiating IIA form (Chambers et al, 1995). Although TFIIB is able to inhibit TFIIF-stimulated Fcpl phosphatase activity, the functional role of this inhibition is still unclear (Chambers et al, 1995). Undoubtedly, the counteracting activity between Ctkl-Fcpl and TFIIH-Ssu72 on CTD phosphorylation must play an important role in initiation, elongation, termination, and transcription-coupled mRNA processing.

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