A: TFIIB Stabilizes TFIID Promoter Binding

Once TFIID is bound to the promoter in the absence or presence of TFIIA, TFIIB is the next GTF to enter the PIC assembly pathway. Binding of TFIIB to promoter-bound TFIID results in a more stable ternary complex composed of TFIID-TFIIB-DNA (Orphanides et al., 1996). Besides stabilizing TFIID binding to the promoter region, TFIIB plays an important role in recruiting pol II/TFIIF to the TFIID-TFIIB-DNA ternary complex and in specifying the transcription start site (Orphanides et al., 1996; Hampsey, 1998). In humans, TFIIB exists as a single 33-kDa polypeptide (Ha et al., 1991; Malik et al., 1991), which shares sequence homology with a 38-kDa Drosophila TFIIB protein and also with a 38-kDa yeast protein encoded by the SUA7 gene (Pinto et al., 1992; Wampler and Kadonaga, 1992; Yamashita et al., 1992). TFIIB is conserved among humans, Drosophila, and yeast, and shows conservation at the N-terminal zinc-ribbon motif and the C-terminal domain containing two imperfect direct repeats (Orphanides et al., 1996; Hampsey, 1998). These two functional domains were originally identified through protease digestion, as the N-terminus is rapidly degraded leaving behind a protease-resistant C-terminal "core" that retains the two imperfect direct repeats of TFIIB (Barberis et al., 1993; Malik et al., 1993).

B: TFIIB-TBP-Promoter Structure

The C-terminal "core" of TFIIB comprises nearly two-thirds of the protein and contains two imperfect repeats each consisting of 5-helices (Bagby et al., 1995). The crystal structure of the TFIIB C-terminal "core", as revealed in a TATA-TBP-TFIIB ternary complex, indicates that TFIIB bound beneath and to one face of the TATA-TBP complex, consistent with footprinting and cross-linking experiments (Coulombe et al., 1994; Lee and Hahn, 1995; Nikolov et al, 1995; Lagrange et al., 1996). Structural evidence also exists for TFIIB competing with negative cofactor 2 (NC2) for overlapping binding sites on TBP (Kamada et al., 2001b). The conserved C-terminus of TFIIB interacts with both TBP and DNA, making contacts with DNA sequences on both sides of the TATA box (Nikolov et al., 1995; Tsai and Sigler, 2000). Experiments also indicate that TFIIB can make sequence-specific DNA contacts with a TFIIB-response element (BRE) typically located just upstream of some TATA box sequences via a helix-turn-helix motif which stabilizes the ternary TFIIB-TBP-promoter complex (Lagrange et al., 1996; Evans et al., 2001; Wolner and Gralla, 2001). Recent studies also show that TFIIB may make sequence-specific DNA contacts with a BRE-like element situated downstream of the TATA box, and that these TFIIB-DNA interactions modulate TFIIB conformation

(Fairley et al., 2002). Moreover, binding of the transcriptional activator Gal4-VP16 to TFIIB induces conformational changes on TFIIB and weakens TFIIB interaction with the BRE (Evans et al., 2001). The role of activator in disrupting TFIIB-DNA interactions seems contradictory with the previously proposed role of the BRE in stabilizing the TFIIB-TBP-DNA ternary complex. It was suggested that the BRE may play an inhibitory role in PIC formation and the presence of an activator alleviates BRE-mediated repression and therefore stimulates overall transcription (Evans et al., 2001). Nevertheless, how activators, TFIIB, and BRE interactions affect PIC assembly, initiation, and promoter clearance remains to be investigated.

C: TFIIB N-Terminal Domain

The TFIIB amino terminus contains a zinc ribbon motif that interacts with components of pol II and TFIIF, and thus facilitates the recruitment of pol II/TFIIF to the TFIID-bound promoter region (Buratowski and Zhou, 1993; Ha et al., 1993; Malik and Roeder, 1993; Yamashita et al., 1993; Fang and Burton, 1996; Bangur et al., 1997). The N- and C-termini of TFIIB engage in an intramolecular interaction that undergoes an activator-induced conformational change, which frees up the N-terminal domain for the recruitment of pol II/TFIIF (Glossop et al., 2004). Immediately adjacent to the N-terminal zinc ribbon motif is a highly conserved region called the charged cluster domain (CCD) or B-finger, which contains key charged amino acid residues. It is believed that the CCD acts as a molecular switch to regulate the conformational change of TFIIB, thereby modulating the role of TFIIB in promoter recognition, start site selection, and transcriptional activation (Pinto et al, 1994; Pardee et al., 1998; Hawkes and Roberts, 1999; Wu and Hampsey, 1999; Hawkes et al., 2000; Faitar et al., 2001; Elsby and Roberts, 2004).

D: CCD-Induced Conformational Changes

As mentioned previously, TFIIB undergoes a conformational change when it interacts with DNA or activators and that the CCD plays a vital role in this conformational change (Fairley et al., 2002; Elsby and Roberts, 2004). The activator Gal4-VP16 has been shown to disrupt the intramolecular interaction between the CCD and the second repeat of the C-terminal domain (Elsby and Roberts, 2004). Corresponding evidence with mutations in the CCD, which favors N-and C-terminal intramolecular interactions, also shows defects in activator-mediated recruitment and in transcriptional activation in vivo and in vitro (Hawkes et al., 2000; Glossop et al., 2004; Elsby and Roberts, 2004). These CCD mutations, however, are competent in PIC assembly, indicating that mutations in the CCD do not affect TFIIB interactions with TBP/DNA and pol II/TFIIF, mediated individually by the C-terminal core and the zinc ribbon motif. Related studies in yeast TFIIB also suggest that conformational changes in TFIIB regulate the stability of the TFIIB-TBP-promoter ternary complex (Bangur et al., 1999).

E: Role of CCD in Start Site Selection

The effect of yeast TFIIB (Sua7) on start site selection has been observed on CYC1, ADH1, and many other genes (Pinto et al., 1992; Berroteran et al., 1994). Besides its role in mediating activated transcription, the CCD also has a critical role in directing accurate initiation of transcription. The functions of the CCD in transcriptional activation and start site selection are located on different amino acid residues (Hawkes and Roberts, 1999). Similar to the activation-defective CCD mutants, distinct mutations in the CCD with aberrant transcriptional start site selection also do not show defects in PIC assembly (Hawkes and Roberts, 1999; Fairley et al., 2002). It has been proposed that these distinct CCD mutations, which cannot undergo proper conformational changes, alter TFIIB's interaction with the pol II catalytic center, thus shifting the transcription start site. Recent photocrosslinking experiments have shown that the N-terminal zinc ribbon interacts with the pol II dock domain and that TFIIB helps position the path of promoter DNA across the central cleft of pol II (Chen and Hahn, 2003, 2004). Supporting crystallographic data has also shown that the CCD of TFIIB forms a finger-like structure that projects into the active center of pol II (Bushnell et al., 2004). Although mutations in the CCD may not be directly involved in PIC formation, conformational changes in TFIIB does play an essential role in transcriptional activation, promoter recognition, and also start site selection.

0 0

Post a comment