presumably via the same region in o that shares homology with RAP30 (McCracken and Greenblatt, 1991). The solution structure of the C-terminal 86 amino acid residues of RAP30, revealed by multinuclear NMR spectroscopy, shows that the DNA-binding domain of RAP30 belongs to the eukaryotic "winged" helix-turn-helix (HTH) family of DNA-binding domains, similar to that found in histone H5 and the hepatocyte nuclear transcription factor HNF-3y (Groft et al., 1998).

The larger subunit of TFIIF, RAP74 (calculated 58 kDa, apparent mass by SDS-PAGE -74 kDa), is highly rich in charged, in particular, acidic amino acid residues (Aso et al., 1992; Finkelstein et al., 1992). This highly charged central region, significantly lacking hydrophobic residues, is hypothesized to be externally exposed and unstructured within the PIC (Yong et al, 1998). The N-terminus of RAP74 is a globular region responsible for interaction with RAP30 (Fang and Burton, 1996). It is involved in PIC assembly, initiation and elongation (Lei et al., 1998; Funk et al., 2002). Similar to RAP30, RAP74 also contains a cryptic DNA-binding domain belonging to the winged HTH family, in which the structure spanning the C-terminal 155 amino acid residues of RAP74 has been revealed by X-ray crystallography (Kamada et al., 2001a). The C-terminal region of RAP74 interacts with the FCP1 phosphatase, which removes phosphorylation on serine 2 at the carboxy-terminal domain (CTD) of the pol II RPB1 subunit, and is necessary and sufficient for FCP1 phosphatase activity in vitro (Archambault et al., 1997, 1998). The interaction with FCP1 may account for the requirement of the C-terminal region of RAP74 for multiple rounds of transcription (Lei et al., 1998; see Core Pol II section). As with other GTFs, TFIIF may also be recruited to the promoter through interaction with transcriptional activators. Indeed, the androgen receptor has been shown to functionally interact with both N- and C-terminal domains of RAP74 (McEwan and Gustafsson, 1997; Reid et al., 2002).

C: Multiple Roles of TFIIF

TFIIF plays multiple roles during PIC assembly. First, TFIIF tightly associates with pol II (Sopta et al., 1985). The RAP74 subunit of yeast TFIIF has been shown to contact the dissociable RPB4/RPB7 subunits of yeast pol II, based on structural comparison between pol II-TFIIF and pol II by cryo-electron microscopy (Chung et al., 2003), and to interact with the RPB9 subunit of yeast pol II (Ziegler et al., 2003; Ghazy et al., 2004). Human RAP30 has also been shown to interact with the RPB5 subunit of human pol II (Wei et al., 2001). The interaction between TFIIF and pol II facilitates the recruitment of pol II to the promoter bound TFIID-TFIIB complex (Flores et al., 1991). Second, TFIIF serves as a stability factor to enhance the affinity of pol II for the TFIID-TFIIB-promoter complex by providing additional protein-DNA contact surfaces and also by inducing changes in DNA topology which causes the promoter to wrap around pol II (Robert et al., 1998). This TFIIF-induced conformational change creates a stable TFIID-TFIIB-pol II-TFIIF-promoter DNA complex that is likely to confer resistance to inhibition by transcriptional repressors that target PIC assembly to negatively regulate gene transcription (Hou et al., 2000). In the study of human papillomavirus (HPV) E2-mediated transcription repression, we found that E2 could still inhibit HPV transcription when added to a preformed TFIID-TFIIB-pol II-promoter DNA complex; however, E2 failed to inhibit transcription once a TFIID-TFIIB-pol II-TFIIF-DNA complex was formed, presumably a surface targeted by E2 for repression was masked by TFIIF-induced DNA wrapping on this minimal PIC (Hou et al., 2000). Third, TFIIF is necessary for subsequent recruitment of TFIIE and TFIIH

(Orphanides et al., 1996) likely via direct interactions with TFIIE (Maxon et al., 1994). Fourth, TFIIF, together with pol II and TFIIB, plays a role in transcription start site selection (Fairley et al., 2002; Ghazy et al, 2004). Fifth, TFIIF has also been implicated in aiding pol II promoter escape (Yan et al., 1999). Following promoter escape, TFIIF then enhances the efficiency of pol II elongation (Shilatifard et al., 2003). Lastly, TFIIF increases the specificity and efficiency of polymerase transcription, similar to bacterial o factors, by preventing spurious initiation through inhibiting and/or reversing the binding of pol II to nonpromoter DNA sequences (Orphanides et al., 1996; Hampsey, 1998). It is clear that TFIIF has multiple roles in the pol II transcription process.

Core Pol II

A: Subunit Composition

Pol II is the key catalytic enzyme in the PIC responsible for transcription of protein-coding genes in eukaryotes. Yeast and human pol II both contain 12 subunits, designated RPB1 to RPB12 by decreasing order of their molecularmass (Young, 1991). In general, the 12 subunits of pol II are highly conserved in sequence, architecture, and function. Indeed, seven subunits of human pol II can either partially (RPB4, RPB7, and RPB9) or completely (RPB6, RPB8, RPB10, and RPB12) substitute for the function of their yeast counterparts in complementation assays (McKune et al., 1995; Khazak et al., 1998). Of the 12 pol II subunits, five (RPB5, RPB6, RPB8, RPB10, and RPB12) are commonly shared among RNA polymerase I, II and III (Woychik et al., 1990; Carles et al., 1991; Young, 1991; Hampsey, 1998). Four pol II subunits, RPB1, RPB2, RPB3, and RPB11, have sequence-homologous counterparts in RNA polymerase I and III. Only RPB4, RPB7, RPB9 and the carboxy-terminal domain (CTD) of RPB1 are unique to pol II. In addition, RPB1, RPB2, RPB3, and RPB6 share similar primary sequences with bacterial RNA polymerase subunits P', P, a, and co respectively (Tan et al., 2000; Minakhin et al., 2001; Mitsuzawa and Ishihama, 2004). A prokaryotic a-like sequence also exists in RPB11 (Woychik et al., 1993; Ulmasov et al., 1996). The primary sequence similarity between RPB1 and P' as well as between RPB2 and P also corresponds to functional similarity: RPB1 and P' are involved in DNA binding, while RPB2 and P bind nucleotide substrates (Hampsey, 1998). Analogous to their bacterial counterparts, RPB1 and RPB2 are responsible for most of the catalytic activity of polymerase and are essential for phosphodiester bond formation (Hampsey, 1998; Lee and Young, 2000).

B: Structure of Pol II

Recently there has been a wealth of structural information on prokaryotic and eukaryotic RNA polymerases provided by photocrosslinking, X-ray crystallography, NMR, and cryo-electron microscopy. The structures for a yeast pol II 10-subunit enzyme minus RPB4 and RPB7 in the absence of DNA (Cramer et al., 2000, 2001) and for a complete 12-subunit pol II have recently been resolved by X-ray crystallography (Armache et al., 2003, 2005; Bushnell and Kornberg,

2003). Initially, RBP4 and RBP7 were not included in the original crystals since the heterodimeric RPB4/ RPB7 module is found in substoichiometric amounts in pol II and may dissociate from the "core" 10 subunits. Furthermore, the RPB4/RPB7 heterodimer, although they are required for PIC formation and initiation of transcription, are dispensable for RNA chain elongation (reviewed by Hampsey, 1998; Lee and Young, 2000; Cramer, 2004; Hahn, 2004). The structure of the 12-subunit pol II complex pinpoints the location of RPB4/RPB7 close to the RNA exit channel and also suggests a role of this heterodimer in transcriptional initiation (Armache et al., 2003, 2005; Bushnell and Kornberg, 2003). Comparison of the structures between the 10-subunit free core enzyme and a transcribing elongation complex containing the same core enzyme in complex with 9 base pairs of an RNA-DNA hybrid within a partially unwound DNA duplex has revealed detailed information regarding subunit-subunit and protein-nucleic acid contacts both within and outside the catalytic center of the enzyme (Cramer et al., 2001; Gnatt et al., 2001). Furthermore, contact residues between pol II and distinct domains of TFIIB, based on the structural information, have also been defined by photocrosslinking experiments (Chen and Hahn, 2003,

2004). The structures of pol II complexed with TFIIB (Bushnell et al., 2004), with elongation factor IIS (Kettenberger et al., 2003), and with IIS in the presence of NTPs and a transcription bubble-mimicking DNA-RNA hybrid (Kettenberger et al., 2004) have also been elucidated by X-ray crystallography. Low-resolution cryo-electron microscopy has resolved structures for a pol II-Mediator complex (Davis et al., 2002) and also for pol II interaction with TFIIF (Chung et al., 2003). The implications of these structural studies have been the focus of many recent reviews (Woychik and Hampsey, 2002; Asturias, 2004; Cramer, 2004; Hahn, 2004; Boeger et al., 2005).

C: CTD Phosphorylation

An essential feature of all pol II complexes resides in the carboxy-terminal domain (CTD) of RPB1, the largest subunit of pol II. The CTD of RPB1 contains a tandem repeat of a heptapeptide: Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Lee and Young, 2000), which repeats 52 times in humans, 42 times in Drosophila, and 26 to 29 times in yeast depending upon the species (Hampsey, 1998; Lee and Young, 2000). The CTD is unstructured and tends to be degraded by proteases. Depending on the phosphorylation state and the presence or absence of the CTD, three forms of pol II (IIO, IIA, and IIB) can be easily distinguished (Kershnar et al., 1998) (Fig. 2.3). The IIA form of pol II contains a hypo- or unphosphorylated form of CTD normally implicated in PIC assembly and transcription initiation. The IIO form of pol II, involved in transcript elongation and termination, has a highly phosphorylated CTD with phosphorylation occurring primarily at serine residues 2 and 5. The IIB form of pol II does not have the CTD but remains transcriptionally active for at least the adenovirus major late promoter (Kang and Dahmus, 1993).

Several protein kinases implicated in CTD phosphorylation have been identified in humans and include cyclin-dependent kinase 7 (CDK7) associated with TFIIH, CDK8 found in general cofactor Mediator, and CDK9 present in positive transcription elongation factor b (P-TEFb). The activities of these CTD kinases are regulated by their associated cyclins that form CDK7-cyclin H, CDK8-cyclin C, and CDK9-cyclin T pairs. Similar CTD kinases have also been identified in yeast and include Cdk7/Kin28, Cdk8/Srbl0, the CTD kinase 1 (CTDK-I), and Sgvl/Burl (Prelich, 2002). Both Burl and the catalytic subunit of CTDK-I, Ctkl, show sequence homology to mammalian CDK9. Phosphorylation of serine 5 by Cdk7/Kin28 following PIC assembly leads to the initiation of transcription and later recruitment of mRNA-capping enzyme guanylyltransferase (Cho et al., 1997; Komarnitsky et al., 2000; Rodriguez et al., 2000; Schroeder et al., 2000; Pei et al., 2001). Phosphorylation of serine 2 by other CTD kinases, such as CTDK-I or P-TEFb (Cho et al., 2001; Zhou et al., 2000; Shim et al., 2002), leads to transcription-coupled recruitment of splicing and 3'-end processing factors (Komarnitsky et al., 2000; Ahn et al., 2004). Two other CTD kinases, Cdk8/Srbl0 (Hengartner et al., 1998) and c-Abl (Baskaran et al., 1993), has also been implicated in phosphorylation of serine 2 and tyrosine 1, respectively, although the functional effects remain to be further defined.


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