TFIIA interactions

Finally, in Plate 21, the structure of the yeast TFIIA-TBP-DNA complex is shown. TFIIA interacts with TBP and the TFIID complex and stimulates transcription of the Pol II gene.29-32 TFIIA has two characteristic structural motife. One is a six-stranded (i-sand-wich, the other is a left-handed four-helix bundle. The P-sandwich domain of TFUA is alone responsible for all of the interactions with the DNA, whereas its helix-bundle domain projects away and is free to interact with signal-responsive transcription factors. These interactions are important for regulation of transcription. Little conformational change occurs in TBP when it binds to TFIIA. The main difference between the TFIIA-TPB complex and the TFIIB-TBP complex is that TFIIA binds upstream of the TATA box, away from the transcriptional start site, whereas TFIIB binds downstream of the TATA box. Moreover, TFIIB is positioned on the side opposite to TFIIA. 

Figure 28.21. Assembly of the Initiation Complex. A ternary complex between the TATA-box-binding protein (purple), TFIIA (orange), and DNA. TFIIA interacts primarily with the other protein.

TFIIA interacts directly with TBP and also with TAF11 (Kraemer et al., 2001). The interaction stabilizes the TFIID-promoter DNA complex and may induce a conformational change in TFIID that facilitates further PIC assembly (Chi and Carey, 1996). TFIIA is also a target of activators and plays an important role against many of the following inhibitory mechanisms. 

TFIIA and TFIIB are two basal transcription factors that are involved in the nucleation stages of the preinitiation complex by binding to the TBP-TATA box complex. Crystal structures of the ternary complex TFIIA-TBP-TATA box have been determined by the groups of Paul Sigler, Yale University, and Timothy Richmond, ETH, Zurich, and that of the TFIIB-TBP-TATA box by Stephen Burley and collaborators. The TBP-DNA interactions and the distortions of the DNA structure are essentially the same in these ternary complexes as in the binary TBP-TATA complex. 

TFIIA also has two domains, one of which is a four-helix bundle and the other an antiparallel p sandwich. The p sandwich interacts with the N-termi-nal half of TBP and thus positions TFIIA on the other side of the complex compared with TFIIB. This domain also interacts with phosphates and sugars of DNA upstream of the TATA box. Tbe four-helix bundle domain makes no contact with DNA or TBP and is far removed from the position of TFIIB. 

Sequences farther upstream from the start site determine how frequently the transcription event occurs. Mutations in these regions reduce the frequency of transcriptional starts tenfold to twentyfold. Typical of these DNA elements are the GC and CAAT boxes, so named because of the DNA sequences involved. As illustrated in Figure 37—7, each of these boxes binds a protein, Spl in the case of the GC box and CTF (or C/EPB,NF1,NFY) by the CAAT box both bind through their distinct DNA binding domains (DBDs). The frequency of transcription initiation is a consequence of these protein-DNA interactions and complex interactions between particular domains of the transcription factors (distinct from the DBD domains—so-called activation domains ADs) of these proteins and the rest of the transcription machinery (RNA polymerase II and the basal factors TFIIA, B, D, E, F). (See... 

Figure 37-9. The eukaryotic basal transcription complex. Formation of the basal transcription complex begins when TFIID binds to the TATA box. It directs the assembly of several other components by protein-DNA and protein-protein interactions. The entire complex spans DNA from position -30 to +30 relative to the initiation site (+1, marked by bent arrow). The atomic level, x-ray-derived structures of RNA polymerase II alone and ofTBP bound to TATA promoter DNA in the presence of either TFIIB or TFIIA have all been solved at 3 A resolution. The structure of TFIID complexes have been determined by electron microscopy at 30 A resolution. Thus, the molecular structures of the transcription machinery are beginning to be elucidated. Much of this structural information is consistent with the models presented here.

RNA polymerases interact with unique cw-active regions of genes, termed promoters, in order to form preinitiation complexes (PICs) capable of initiation. In eukaryotes the process of PIC formation is facilitated by multiple general transcription factors (GTFs), TFIIA, B, D, E, F, and H. 

In vitro interactions between HMG proteins and the basal transcription machinery have also been reported. Human HMGBl binds to the TATA-box binding protein (TBP) and interferes with the normal binding of TFIIB in the preinitiation complex [154,155], thereby inhibiting TBP function both HMGBl and TFIIB independently enhance binding of TBP to TATA-box DNA [154]. Similarly, Nhp6ap promotes the formation of a complex with TBP and TFIIA at the TATA... 

TFIIA and TFIIB support TFIID in the formation of a stable complex with the promotor. TFllB is necessary for the downstream selection of the start site for RNA polymerase 11. Interactions with TFllB ensure correct positioning of the RNA polymerase 11 on the promoter. Crystal structures have been solved for several of the intermediates of the pre-initiation complex (review Sokolev and Burley, 1997), showing, for example, that TBP affects a predominant kink in the DNA (see Fig. 1.16). TFIIB binds to the TBP-DNA complex, contacting both TBP and the DNA. 

Interaction of the TATA-box-binding protein (TBP) with promoter DNA is rather inefficient and appears to be the rate-limiting step for the start of transcription. TBP must actually dissociate first from the TFIID complex before it can bind to the TATA-box DNA. Dissociation of TBP is facilitated by the dimeric structure of TFIID, when it is not bound to DNA, and by the interaction of TBP with TFIIA. 

We have learnt from the structures that the TBP/TATA complex is a nucleoprotein scaffold upon which other factors, such as TFIIA and TFIIB attach with high affinity through a combination of stereospecific and electrostatic interactions. TFIIB is positioned at the transcriptional start site, between the attachment sites for TBP and Pol II. On the other hand, TFIIA has no contacts with the DNA downstream of the TATA box and does not interact with the transcriptional start site and/or any of the components of the basal transcription machinery, all of which are located downstream of the TATA box. Thus, TFIIA and TFIIB can bind simultaneously, without mutual interference. Moreover, TFIIA is accessible to specific, signal-responsive regulatory transcription factors. Its location upstream of the TATA box also enables TFIIA to absorb and scavenge transcriptional inhibitors, making them ineffectual. 

The entire top side of TBP bound to the TATA-box DNA is out of the way and is fi e to interact with other factors. This leaves a generous surface on TBP available for interactions with the multitude of factors which are parts of the transcription initiation complex. The same is true for the TFIIA/TFIIB-TBP-complexes. The extensive surfaces displayed by the TFIIA/TFIIB-TBP-DNA complexes represent potential sites for binding basal initiation factors, signal-responsive transcriptional activators, co-activators and mediators, and, most importantly, leave room for Pol II. 

Ternary complexes formed by TBP, DNA and TFIIA or TFIIB have also been crystallized [27-32] and their structures are available in the PDB and NDB (see Table 1). These complexes display practically the same mode of interaction between TBP and the DNA moiety as found in the corresponding binary complexes. They also explain the inability of TFIIB to bind to DNA on its own, as it is found to contact the DNA upstream and downstream of TBP, an impossible feat in a straight DNA molecule. This feature places TBP among the architectural transcription factors, together with DBF, HMG, SRY and LEFl... 

As shown in Figure 11.19, once TFIID is bound, TFIIA binds, and TFIIA also interacts with both the DNA and TFIID. TFIIB also binds to TFIID, bridging the TBP and Pol 11. TFIIA and TFIIB can bind in either order, and they do not interact with each other. TFIIB is critical for the assembly of the initiation complex and for the location of the correct transcription start site. TFIIF then binds tightly to Pol 11 and suppresses nonspecific binding. Pol II and TFIIF then bind stably to the promoter. TFIIF interacts with Pol II, TBP, TFIIB, and the TAFIIs. It also regulates the activity of the GTD phosphatase. 

Fig. 9-4 Promoters interact with distant enhancers via iooping, faciiitated by the mediator compiex. (A) DNA iooping around so its enhancer sequences, via activator transcription factors, interact with the compiex that forms around RNA poiymerase ii. (B) RNA poiymerase ii, iike other eukaryotic RNA poiymerases, is recruited by transcription factors which assembie step-wise at the promoter, its C terminai taii is phosphoryiated to recruit eionga-tion factors which permit it to ieave the promoter and commence transcription. The various transcription factors (TFiiA, TFiiB, etc.) are indicated by their abbreviated names, uniike FiAT which is histone acetyi transferase.

TFIIB interacts with the C-terminal stirrup of TBP and the DNA both upstream and downstream of the TATA box, whereas TBIIA interacts with the N-terminal stirrup and the DNA upstream of the TATA box on the opposite face of the double helix from TFIIB (Nikolov and Burley, 1997). Therefore, both TFIIA and TFIIB can bind to the TBP-DNA complex simultaneously and synergistically stabilize the complex. Furthermore, TFIIB binding also contributes to the directionality of transcription and forms a bridge between TBP and Pol II and specifies the transcription start site. It will be interesting to see how these interactions fit into the structure of the TFIID complex. 

In the context of TFIID, a TAF can also function as an inhibitor of TBP-DNA interactions. The N-terminus of TAF1 has been found to interact with TBP and block TBP-DNA interaction by mimicking the surface of partially unwound minor groove of TATA box when bound to TBP (Kokubo et al., 1993 Liu et al., 1998). This inhibitory interaction is counteracted by TFIIA and by c-Jun, a transcription activator (Kokubo et al., 1998 Lively et al., 2001). 

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